acid-phosphatase and tartaric-acid

Topics: Acid Phosphatase; Biomarkers; Clinical Enzyme Tests; Diagnosis, Differential; Hematologic Diseases; Hematologic Tests; Humans; Leukemia, Hairy Cell; Leukocytes; Reference Values; Specimen Handling; Tartrates

A review is given summarizing the present knowledge of bone turnover markers with special emphasis on biological, preanalytical and technical criteria in the proper judgement of efficacy and limitations of the methods employed. The marker substances may be either measures of bone formation or bone resorption. Markers of bone formation are bone alkaline phosphatase, osteocalcin and the carboxyl-terminal propeptide of procollagen type I. Bone alkaline phosphatase has proved to be superior to total alkaline phosphatase activity with respect to diagnostic sensitivity and specificity. Immunochemical techniques for measuring bone alkaline phosphatase show a cross-reactivity of 14-20% with liver alkaline phosphatase. However, this does not compromise the clinical usefulness of these assays except for patients with severe liver diseases. Osteocalcin is strictly bone-specific but shows numerous disadvantages with respect to apparent instability and discordant results as obtained by different methods; however, in certain diagnostic situations (corticosteroid-induced osteopenia, absence of destroyed bone architecture) osteocalcin may serve as a sensitive bone turnover marker. The carboxyl-terminal propeptide of procollagen type I generally shows low discriminating power in the diagnosis of bone diseases. The urinary excretion of pyridinium "cross-links' has been carefully evaluated so far with respect to analytical performance and clinical usefulness. This marker may be a substitute for 4-hydroxyproline measurements as the method of choice for assessment of bone resorption. There are other degradation products from the telopeptide regions of bone-derived collagen type I which are excreted into the urine (N-telopeptides, CrossLapsTM); these analytes are promising tools in the assessment of bone resorption but require further evaluation, in particular with respect to their extraskeletal clearance and putative origin outside bone. Moreover, their clinical usefulness may vary depending on the patient group examined. In contrast, the serum concentration of the cross-linked telopeptide region of collagen type I seems to lack both diagnostic specificity and sensitivity in the majority of patient groups. Tartrate-resistant acid phosphatase (as determined by the presently available methods) cannot be recommended as a routine tool for assessment of bone resorption.

Topics: Acid Phosphatase; Adult; Alkaline Phosphatase; Amino Acid Sequence; Animals; Biomarkers; Bone Diseases; Bone Remodeling; Collagen; Collagen Type I; Female; Humans; Male; Menopause; Molecular Sequence Data; Osteocalcin; Osteonectin; Peptides; Pyridinium Compounds; Sialoglycoproteins; Tartrates

Topics: Acid Phosphatase; Cytogenetics; Diagnosis, Differential; Humans; Immunologic Techniques; Leukemia, Hairy Cell; Microscopy, Electron; Tartrates

The increase in acid phosphatase (AP) activity in cultured canine prostatic epithelial cells was investigated as a biochemical marker of in vitro cellular differentiation. The enzyme was studied in secretory and non-secretory epithelial cell populations obtained from control and cycloheximide-treated cultures over a period of 3 weeks and compared to the AP present in tissue and cellular extracts from normal canine prostates. The progressive increase in AP activity with the duration of culture was strongly inhibited by cycloheximide in both cell populations. The degree of inhibition was more pronounced late in the culture when AP activity increased at a faster rate in secretory cells. Cycloheximide inhibited protein biosynthesis by 70-80% as evidenced by a reduction in the incorporation of amino acids into acid-insoluble material. However, the specific activities of AP in the cellular extracts were similar in control and cycloheximide-treated cultures and increased sharply by 3-4-fold in the secretory cells after 12 days of culture. When extracts derived from control and cycloheximide-treated cells of various duration were submitted to electrophoresis in polyacrylamide gels (PAGE), a unique pattern of three bands of AP activity with Rf values of 0.18, 0.27 and 0.38 was obtained. In controls the AP activity in the band with an Rf of 0.18 increased preferentially during the culture period and was more important quantitatively in secretory cells. In cycloheximide-treated cultures the increase of AP activity associated with the band with an Rf of 0.18 was more strongly inhibited. The addition of tartrate to the staining mixture inhibited all three bands of AP activity. Similar results were obtained when extracts derived from freshly dispersed cells as well as from normal canine prostatic tissue were submitted to PAGE; the AP activity was resolved into 3 bands with Rf values of 0.15-0.18, 0.23-0.27 and 0.33-0.38; all three bands were inhibited by the addition of tartrate and the first band was predominant. Thus, the increase in AP activity in prostatic epithelial cells in a culture medium supplemented with serum and deprived of sex steroids is due to the de novo synthesis of a major form of the enzyme by the secretory cells.

Topics: Acid Phosphatase; Animals; Cell Differentiation; Cells, Cultured; Cycloheximide; Dogs; Male; Prostate; Tartrates

Topics: Acid Phosphatase; Adenine; Amino Acid Sequence; Apoenzymes; Catalytic Domain; Histidine; Legionella pneumophila; Models, Molecular; Mutation; Phenylalanine; Protein Binding; Ribose; Sequence Alignment; Substrate Specificity; Tartrates

The effects of sodium citrate (SC), sodium acetate (SA) and sodium tartrate (ST) spraying on mung bean germination were investigated. Exogenous SC, ST and SA treatments significantly reduced the phytic acid content and increased the antioxidant enzyme activities. In this study, an iTRAQ-based proteomic approach was employed to explore the proteomes of mung bean sprouts, and 81, 101 and 90 differentially expressed proteins were identified in 4-day-old SC-, SA- and ST-treated mung bean sprouts, with 38 proteins present in all samples. Functional classification analysis showed that most of the differentially expressed proteins in mung bean sprouts subjected to the three treatments were involved in carbohydrate and energy metabolism. The inhibitory effect of the SA treatment was probably due to impairments in protein biosynthesis, whereas enhanced energy metabolism, accelerated reserve hydrolysis and protein processing were very important strategies for growth stimulation in response to ST and SC treatments.

Topics: Acid Phosphatase; Citrates; Food Additives; Food Handling; Gene Expression Regulation, Plant; Glycoproteins; Inositol Polyphosphate 5-Phosphatases; Phytic Acid; Plant Proteins; Protein Biosynthesis; Proteomics; Sodium Acetate; Sodium Citrate; Tartrates; Up-Regulation; Vigna

Histidine acid phosphatases (HAPs) utilize a nucleophilic histidine residue to catalyze the transfer of a phosphoryl group from phosphomonoesters to water. HAPs function as protein phosphatases and pain suppressors in mammals, are essential for Giardia lamblia excystation, and contribute to virulence of the category A pathogen Francisella tularensis. Herein we report the first crystal structure and steady-state kinetics measurements of the HAP from Legionella pneumophila (LpHAP), also known as Legionella major acid phosphatase. The structure of LpHAP complexed with the inhibitor l(+)-tartrate was determined at 2.0 Å resolution. Kinetics assays show that l(+)-tartrate is a 50-fold more potent inhibitor of LpHAP than of other HAPs. Electrostatic potential calculations provide insight into the basis for the enhanced tartrate potency: the tartrate pocket of LpHAP is more positive than other HAPs because of the absence of an ion pair partner for the second Arg of the conserved RHGXRXP HAP signature sequence. The structure also reveals that LpHAP has an atypically expansive active site entrance and lacks the nucleotide substrate base clamp found in other HAPs. These features imply that nucleoside monophosphates may not be preferred substrates. Kinetics measurements confirm that AMP is a relatively inefficient in vitro substrate of LpHAP.

Topics: Acid Phosphatase; Adenosine Monophosphate; Amino Acid Motifs; Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Escherichia coli; Gene Expression; Histidine; Kinetics; Legionella pneumophila; Models, Molecular; Molecular Sequence Data; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Static Electricity; Substrate Specificity; Tartrates

High-scale purification methods are required for several protein studies such as crystallography, mass spectrometry, circular dichroism, and function. Here we describe a purification method for PAP based on anion exchange, L-(+)-tartrate affinity, and gel filtration chromatographies. Acid phosphatase activity and protein concentration were measured for each purification step, and to collect the fractions with the highest acid phosphatase activity the p-nitrophenyl phosphate method was used. The purified protein obtained by the procedure described here was used for the determination of the first reported three-dimensional structure of prostatic acid phosphatase.

Topics: Acid Phosphatase; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Humans; Male; Nitrophenols; Organophosphorus Compounds; Prostate; Protein Tyrosine Phosphatases; Substrate Specificity; Tartrates

This study examined the immunoexpression pattern of aquaporin-1 (AQP1), first identified as a water channel protein, in the periodontal ligament of rat molars during experimental tooth movement to clarify its role in periodontal responses in an overloaded model by the insertion of a piece of elastic band. In the control group without any treatment, the cementoblasts and osteogenic cells as well as the vascular endothelial cells showed AQP1 immunoreaction. In the experimental group, hyalinized tissue and intensely AQP1 positive amorphous structures which were identified as degenerated endothelial cells by immunoelectron microscopy, occurred at the compression side on Days 1 and 3. AQP1 immunoreaction came to be stronger in the intact endothelial cells around the hyalinized tissue. The hyalinized tissue had almost disappeared by Day 5 when many macrophages reactive to acid phosphatase activity appeared. The periodontal width on Day 7 became almost the same as that in the control group. These findings indicate that the hyalinized tissue and damaged AQP1 positive endothelial cells are phagocytized by macrophages which have temporally migrated, and suggest that the surviving endothelial cells with intense AQP1 reaction are involved in periodontal regeneration by capillary sprouting.

Topics: Acid Phosphatase; Animals; Aquaporin 1; Cell Movement; Dental Cementum; Dental Pulp; Endothelial Cells; Enzyme Activation; Immunohistochemistry; Macrophages; Male; Microscopy, Electron; Molar; Periodontal Ligament; Phagocytosis; Rats; Rats, Wistar; Tartrates; Tooth Movement Techniques

A recombinant putative acid phosphatase from Thermus thermophilus was expressed and purified from Escherichia coli. The recombinant phosphatase displayed activities in a broad range of temperature, from 40 to 90 degrees C, with optimal temperature at 70 degrees C. In addition, the recombinant enzyme had activities in a wide range of pH, from 3.6 to 9.1, with optimal pH at 6 in acetate buffer and with optimal pH at 6.5 in Hepes buffer. Furthermore, it showed significant thermal stability and still possessed 44% residual activity after 70 degrees C treatment for 15 min. Moreover, the recombinant phosphatase showed broad substrates specificities for monophosphate esters, p-nitrophenyl phosphate (pNPP) being the most preferred substrate, and it was able to resist inhibition by sodium tartrate. Additionally, the recombinant protein formed stable oligomer under partially denatured conditions and required calcium ions for enzymic activity.

Topics: Acid Phosphatase; Escherichia coli; Nitrophenols; Organophosphorus Compounds; Phosphoric Monoester Hydrolases; Recombinant Proteins; Substrate Specificity; Tartrates; Temperature; Thermus thermophilus

Histidine acid phosphatases catalyze the transfer of a phosphoryl group from phosphomonoesters to water at acidic pH using an active-site histidine. The histidine acid phosphatase from the category A pathogen Francisella tularensis (FtHAP) has been implicated in intramacrophage survival and virulence, motivating interest in understanding the structure and mechanism of this enzyme. Here, we report a structure-based study of ligand recognition by FtHAP. The 1.70-A-resolution structure of FtHAP complexed with the competitive inhibitor l(+)-tartrate was solved using single-wavelength anomalous diffraction phasing. Structures of the ligand-free enzyme and the complex with inorganic phosphate were determined at resolutions of 1.85 and 1.70 A, respectively. The structure of the Asp261Ala mutant enzyme complexed with the substrate 3'-AMP was determined at 1.50 A resolution to gain insight into substrate recognition. FtHAP exhibits a two-domain fold similar to that of human prostatic acid phosphatase, consisting of an alpha/beta core domain and a smaller domain that caps the core domain. The structures show that the core domain supplies the phosphoryl binding site, catalytic histidine (His17), and an aspartic acid residue (Asp261) that protonates the leaving group, while the cap domain contributes residues that enforce substrate preference. FtHAP and human prostatic acid phosphatase differ in the orientation of the crucial first helix of the cap domain, implying differences in the substrate preferences of the two enzymes. 3'-AMP binds in one end of a 15-A-long tunnel, with the adenine clamped between Phe23 and Tyr135, and the ribose 2'-hydroxyl interacting with Gln132. The importance of the clamp is confirmed with site-directed mutagenesis; mutation of Phe23 and Tyr135 individually to Ala increases K(m) by factors of 7 and 10, respectively. The structural data are consistent with a role for FtHAP in scavenging phosphate from small molecules present in host macrophage cells.

Topics: Acid Phosphatase; Amino Acid Substitution; Crystallography, X-Ray; Enzyme Inhibitors; Francisella tularensis; Humans; Models, Molecular; Mutant Proteins; Mutation, Missense; Organophosphates; Protein Binding; Protein Structure, Tertiary; Tartrates

Vitellin (VT) is a lipoglycophosphoprotein stored inside the eggs of every oviparous organism during oogenesis. In the blood-sucking bug Rhodnius prolixus, VT is deposited inside growing oocytes together with two acid hydrolases: acid phosphatase (AP) and cathepsin D (CD). Egg fertilization triggers AP activity and VT proteolysis in vivo [Insect Biochem. Mol. Biol. 2002 (32) 847]. Here, we show that CD is the main protease targeting VT proteolysis during egg development. CD activity in total egg homogenates is blocked by the classical aspartyl protease inhibitor, pepstatin A. Surprisingly, AP inhibitors such as NaF, Na+/K+ tartrate, and inorganic phosphate also block VT proteolysis, whereas this effect is not observed when tyrosine phosphatase inhibitors such as vanadate and phenylarsine oxide or an inhibitor of alkaline phosphatases such as levamisole are used in a VT proteolysis assay. NaF concentrations that block isolated AP activity do not affect the activity of partially purified CD. Therefore, a specific repressor of VT proteolysis must be dephosphorylated by AP in vivo. In conclusion, these results demonstrate for the first time that acid hydrolases act cooperatively to promote yolk degradation during egg development in arthropods.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Cathepsin D; Cathepsins; Egg Proteins; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Hydrolases; Insecta; Oocytes; Oogenesis; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Tyrosine Phosphatases; Rhodnius; Sodium; Tartrates; Temperature; Time Factors; Vitellins

The adverse side effects of currently available anti-osteoporotic agents warrant the search for compounds with less toxic effects. In this study, we assessed the phytoestrogenic potentiality of whole aqueous extract of black tea (BTE) in a bilaterally oophorectomized rat model (2.5%, 1 ml/100 g body weight/day for 28 days). Although the supplementation was given for 28 days but, sign of revival of copulation period (estrous stage) from non-receptive diestrous stage was first noticed after 21 days of BTE supplementation in bilaterally oophorectomized rats. This was accompanied by a significant increase in serum estradiol level. To test whether this increase in serum estradiol level could have an influence upon the oophorectomy-induced damage of bone, we assessed marker parameters of bone resorption and osteoclastic activity (tartrate-resistant acid phosphatase), collagen degradation (urinary hydroxyproline), bone loss (bone ash mineral content) and bone breaking strength (bone density). Results indicated that increase in serum estradiol level after BTE supplementation could significantly diminish oophorectomy-induced decaying changes in bone. This study proposes that aqueous BTE may be assessed as a phytoestrogenic compound for prevention against estrogen deficiency-related osteoporotic damages.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Density; Bone Resorption; Calcium; Collagen; Dietary Supplements; Estradiol; Female; Hydroxyproline; Ovariectomy; Rats; Tartrates; Tea

Thromboxan A(2) (TXA(2)) is the main product of arachidonic acid metabolism in activated platelets. Platelet-released supernatants (PRS) can induce osteoclast-like cell formation in murine bone marrow cultures via a cyclooxygenase (COX)/receptor activator of NF-kB-ligand (RANKL)-dependent pathway. Here we investigated a possible linkage between platelet-released TXA(2) and osteoclastogenesis. The stable analog of TXA(2), carbocyclic TXA(2) (CTXA(2)) can induce the formation of tartrate-resistant acid phosphatase positive multinucleated cells in murine bone marrow cultures via a RANKL-dependent pathway and requires the presence of stromal cells. Interestingly, the platelet-released instable TXA(2) does not account for osteoclastogenic effects as: (a) PRS-induced osteoclastogenesis in the presence of the TXA(2) receptor antagonist SQ29548; (b) inhibition of platelet TXA(2) synthesis by indomethacin and acetylsalicylic acid failed to decrease the osteoclastogenic potential of the corresponding supernatants; and (c) CTXA(2)-induced osteoclast-like cell formation independent of indomethacin and the selective COX-2 inhibitor NS398.

Topics: Acid Phosphatase; Animals; Arachidonic Acid; Aspirin; Blood Platelets; Bone Marrow Cells; Bridged Bicyclo Compounds, Heterocyclic; Carrier Proteins; Cell Nucleus; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Hydrazines; Indomethacin; Membrane Glycoproteins; Mice; Nitrobenzenes; Osteoclasts; Prostaglandin-Endoperoxide Synthases; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Stromal Cells; Sulfonamides; Tartrates; Thromboxane A2

Acid phosphatase (AP) activity was detected in 24 h culture media from adult Heligmosomoides polygyrus. Female and male excretion/secretion products showed similar specific activity. For both, the AP had a pH optimum of 4.0 and was inhibited by sodium fluoride, tartaric acid, and sodium orthovanadate. The release of AP by adult worms was significantly inhibited by adverse incubation conditions (temperatures of 20 degrees C and 4 degrees C), known physiological perturbers ( t-butylhydroperoxide and sodium azide), and broad spectrum anthelmintics (albendazole, levamisole, morantel, and ivermectin). These results indicate that the AP activity level in the culture medium may be an indicator of the physiological status of the worms.

Topics: Acid Phosphatase; Albendazole; Animals; Antigens, Helminth; Culture Media; Female; Hydrogen-Ion Concentration; Ivermectin; Levamisole; Male; Morantel; Nematospiroides dubius; Sensitivity and Specificity; Sodium Fluoride; Tartrates; Temperature; Vanadates

The continuous measurement of acid phosphatase (EC 3.1.3.2) activity in serum represents an analytical task not yet sufficiently accomplished.. Introducing two novel substrates-2-chloro-4-nitrophenyl phosphate (CNP-P), which was preferred, and 4-nitronaphthyl-1-phosphate (NN-P)-an alternative assay to measure enzymatic activity was developed and compared with a modification of Hillmann's method (azo coupling of released naphth-1-ol with a diazonium compound). Apart from different substrate concentrations of 2-chloro-4-nitrophenyl phosphate, 4 mmol/l, and naphthyl-1-phosphate (N-P), 8 mmol/l (with Fast Red TR, 5 mmol/l), respectively, following identical conditions were selected: Citrate, 50 mmol/l, pH 5.75; pentane-1,5-diol, 150 mmol/l; tartrate, 60 mmol/l; 37 degrees C.. Whereas intensity and stability of the azo dye unpredictably depend on the albumin concentration of the sample, the direct test with 2-chloro-4-nitrophenyl phosphate resisted sample interferences, showed no intrinsic hydrolysis by albumin, relied on stable reagents and proved superior in sensitivity, precision and ease of handling. In measuring prostatic phosphatase, the proposed procedure closely correlated with Hillmann's method. The preliminary 0.95-reference intervals for adults were 1.2-3.9 kU/l and 5.8-14.8 U/l for total activity, respectively.. The direct assay of the enzyme is suited as an economic, rapid and robust method for mechanized or manual use.

Topics: Acid Phosphatase; Azo Compounds; Glycols; Humans; Hydrogen-Ion Concentration; Male; Naphthalenes; Organophosphorus Compounds; Pentanes; Phosphates; Protein Tyrosine Phosphatases; Reagent Kits, Diagnostic; Reference Values; Reproducibility of Results; Solubility; Spectrophotometry; Substrate Specificity; Tartrates

Tartrate-resistant acid phosphatase (TRAP) of Amoeba proteus (strain B) was represented by 3 of 6 bands (= electromorphs) revealed after disc-electrophoresis in polyacrylamide gels with the use of 2-naphthyl phosphate as a substrate at pH 4.0. The presence of MgCl2, CaCl2 or ZnCl2 (50 mM) in the incubation mixture used for gel staining stimulated activities of all 3 TRAP electromorphs or of two of them (in the case of ZnCl2). When gels were treated with MgCl2, CaCl2 or ZnCl2 (10 and 100 mM, 30 min) before their staining activity of TRAP electromorphs also increased. But unlike 1 M MgCl2 or 1 M CaCl2, 1 M ZnCl2 partly inactivated two of the three TRAP electromorphs. EDTA and EGTA (5 mM), and H2O2 (10 mM) completely inhibited TRAP electromorphs after gel treatment for 10, 20 and 30 min, resp. Of 5 tested ions (Mg2+, Ca2+, Fe2+, Fe3+ and Zn2+), only the latter reactivated the TRAP electromorphs previously inactivated by EDTA or EGTA treatment. In addition, after EDTA inactivation, TRAP electromorphs were reactivated better than after EGTA. The resistance of TRAP electromorphs to okadaic acid and phosphatase inhibitor cocktail 1 used in different concentrations is indicative of the absence of PP1 and PP2A among these electromorphs. Mg2+, Ca2+ and Zn2+ dependence of TRAP activity, and the resistance of its electromorphs to vanadate and phosphatase inhibitor cocktail 2 prevents these electromorphs from being classified as PTP. It is suggested that the active center of A. proteus TRAP contains zinc ion, which is essential for catalytic activity of the enzyme. Thus, TRAP of these amoebae is metallophosphatase showing phosphomonoesterase activity in acidic medium. This metalloenzyme differs from both mammalian tartrate-resistant PAPs and tartrate-resistant metallophosphatase of Rana esculenta.

Topics: Acid Phosphatase; Aluminum; Amoeba; Animals; Calcium Chloride; Chlorides; Edetic Acid; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Inhibitors; Magnesium Chloride; Okadaic Acid; Phosphoric Monoester Hydrolases; Tartrates; Zinc Compounds

Macrophage colony stimulating factor (M-CSF) and osteoclast differentiation factor (ODF) regulate osteoclastogenesis in vivo. Regulation of osteoclast development in vitro by these cytokines has been reported in the present study. Simultaneous addition of ODF and M-CSF during initiation of bone marrow culture inhibited osteoclastogenesis. However, delayed addition of ODF (three days after initiation of the culture) resulted in dramatic increase in phenotypically and functionally mature osteoclast cells. Delayed addition of ODF beyond day three decreased osteoclastogenesis. Further, removal of M-CSF as early as day three inhibited ODF-induced osteoclastogenesis. These studies provided evidence for the importance of co-ordinated regulation of osteoclastogenesis by M-CSF and ODF.

Topics: Acid Phosphatase; Actins; Animals; Bone Marrow; Carrier Proteins; Cells, Cultured; Hematopoietic Stem Cells; Macrophage Colony-Stimulating Factor; Membrane Glycoproteins; Mice; Osteoclasts; Osteogenesis; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Recombinant Proteins; Tartrates; Time Factors

The primitive trypanosomatid pathogen of humans, Leishmania donovani, constitutively expresses a unique externally oriented, tartrate-resistant, acid phosphatase on its surface membrane. This is of interest because these organisms are obligate intracellular protozoan parasites that reside and multiply within the hydrolytic milieu of mammalian macrophage phago-lysosomes. Here we report the identification of the gene encoding this novel L. donovani enzyme. In addition, we characterized its structure, demonstrated its constitutive expression in both parasite developmental forms, and determined the cell surface membrane localization of its translated protein product. Further, we used a variety of green fluorescent protein chimeric constructs as reporters in a homologous leishmanial expression system to dissect the functional domains of this unique, tartrate-resistant, surface membrane enzyme.

Topics: Acid Phosphatase; Amino Acid Sequence; Animals; Binding Sites; Blotting, Southern; Deoxyribonucleases, Type II Site-Specific; DNA, Protozoan; Green Fluorescent Proteins; Humans; Leishmania donovani; Luminescent Proteins; Molecular Sequence Data; Open Reading Frames; Protein Conformation; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; Tartrates

A study was carried out over a 24-month interval to determine if an initial measurement of serum tartrate-resistant acid phosphatase would be predictive of bone mass loss quantified by dual-energy X-ray absorptiometry, as total bone mineral content and total bone mineral content corrected for weight.. Sixty-two women were studied (at onset: mean age 59.7 +/- 8.9 years, 10.8 +/- 8.8 years since menopause; at conclusion: mean age 61.9 +/- 8.8 and 13.0 +/- 8.7 since menopause).. A paired Wilcoxon test showed a small, but significant, increase in weight (P < 0.05) and decrease in height (P < 0.05). Total bone mineral content and total bone mineral content corrected for weight decreased (P < 0.005 and 0.0001, respectively). Serum tartrate-resistant acid phosphatase increased (P < 0.005). Single-regression analysis showed that the per cent bone mass loss observed between the first and second body bone mineral content measurements correlated negatively with the first serum tartrate-resistant acid phosphatase determination (r = -0.62, P < 0.0001). Changes in tartrate-resistant acid phosphatase correlated negatively with changes in total bone mineral content (r = -0.79, P < 0.0001). In a multiple regression analysis of per cent change in bone mass against initially important variables such as age, years since menopause, weight, and tartrate-resistant acid phosphatase, only tartrate-resistant acid phosphatase was significant (P < 0.0001). The sensitivity and specifity of tartrate-resistant acid phosphatase for evaluating bone loss were 86% and 78%, respectively, and the area under the curve was of 0.83 (95% CI 0.71-0.95).. These results show that a simple measurement of serum tartrate-resistant acid phosphatase can help to predict the potential rate of bone mass loss in women.

Topics: Absorptiometry, Photon; Acid Phosphatase; Biomarkers; Bone Density; Female; Humans; Middle Aged; Osteoporosis, Postmenopausal; Regression Analysis; Sensitivity and Specificity; Tartrates

The solution structure and thermal stability of human prostatic acid phosphatase (hPAP) in the absence and in the presence of tartaric acid were studied by Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The temperature dependence of the infrared spectrum and DSC scans indicate that hPAP undergoes thermal unfolding at a temperature between 49.5 and 52.5 degrees C. Binding of tartaric acid does not lead to major changes in the secondary structure of hPAP, however, hPAP with bound tartaric acid shows a significantly increased thermal stability. These results helped to better understand the mechanism of hPAP unfolding at the elevated temperature.

Topics: Acid Phosphatase; Calorimetry, Differential Scanning; Humans; Protein Conformation; Protein Tyrosine Phosphatases; Spectrophotometry, Infrared; Tartrates; Temperature

Acid phosphatase [AP; EC 3.1.3.2], a key enzyme involved in the synthesis of mannitol in Agaricus bisporus, was purified to homogeneity and characterized. The native enzyme appeared to be a high molecular weight type glycoprotein. It has a molecular weight of 145 kDa and consists of four identical 39-kDa subunits. The isoelectric point of the enzyme was found at 4.7. Maximum activity occurred at 65 degrees C. The optimum pH range was between 3.5 and 5.5, with maximum activity at pH 4.75. The enzyme was unaffected by EDTA, and inhibited by tartrate and inorganic phosphate. The enzyme exhibits a Km for p-nitrophenylphosphate and fructose-6-phosphate of 370 microM and 3.1 mM, respectively. A broad substrate specificity was observed with significant activities for fructose-6-phosphate, glucose-6-phosphate, mannitol-1-phosphate, AMP and beta-glycerol phosphate. Only phosphomonoesters were dephosphorylated. Antibodies raised against the purified enzyme could precipitate AP activity from a cell-free extract in an anticatalytic immunoprecipitation test.

Topics: Acid Phosphatase; Adenosine Monophosphate; Agaricus; Chemical Fractionation; Chromatography, Gel; Chromatography, Ion Exchange; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Fructosephosphates; Fungal Proteins; Glucose-6-Phosphate; Glycerophosphates; Glycoproteins; Isoelectric Point; Mannitol Phosphates; Molecular Weight; Nitrophenols; Organophosphorus Compounds; Phosphates; Polyethylene Glycols; Precipitin Tests; Protein Subunits; Substrate Specificity; Tartrates; Temperature

An acid phosphatase (AP) and a phosphorylcholine hydrolase (PCH) were detected in excretory-secretory (ESP) products from adult Haemonchus contortus. The AP had a pH optimum of 4.5 and was inhibited by tartaric acid and sodium fluoride, but not by o-phenanthroline. The AP hydrolyzed paranitrophenol (pnp)-phosphate and to a lesser extent pnp-phenyl-phosphonate but did not hydrolyze diester substrates. Purified AP consisted of heterodimers with relative molecular weight (Mr) of 41.9 and 48.7 kDa and had a native molecular weight of 98 kDa by size-exclusion chromatography (SEC). The PCH had a pH optimum of about 9.5 and was inhibited by EDTA and o-phenanthroline but not by the specific phospholipase inhibitor D609. The specific activity of PCH in the ESP was approximately 25-fold less than that of AP. PCH also hydrolyzed 5'-thymidine monophosphate-pnp at a rate about 40% lower than pnp-phosphorylcholine but did not hydrolyze 3'-thymidine monophosphate-pnp. Partial purification of PCH suggests an Mr of 50.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an Mr of 102 kDa by SEC. Both AP and PHC were secreted in vitro in a time-dependent manner and had their highest concentrations in the intestine. The results indicate that H. contortus adults secrete significant amounts of AP that might be a digestive enzyme. PCH is also an intestinal enzyme and is secreted in lesser amounts than AP. The PCH is probably not a phospholipase C but has some characteristics of a type I phosphodiesterase.

Topics: Acid Phosphatase; Animals; Cholinesterase Inhibitors; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Female; Haemonchus; Hydrogen-Ion Concentration; Hydrolases; Hydrolysis; Molecular Weight; Phenanthrolines; Sheep; Sodium Fluoride; Tartrates; Vanadium

The biochemical properties and protein structure of the tartrate-resistant acid phosphatase (TRAP), an iron-containing lysosomal glycoprotein in cells of the mononuclear phagocyte system, are well known. In contrast, little is known about the physiology and genic structure of this unique enzyme. In some diseases, like hairy cell leukemia, Gaucher's disease and osteoclastoma, cytochemically detected TRAP expression is used as a disease-associated marker. In order to begin to elucidate the regulation of this gene we generated different deletion constructs of the TRAP 5'-flanking region, placed them upstream of the luciferase reporter gene and assayed them for their ability to direct luciferase expression in human 293 cells. Treatment of these cells with the iron-modulating reagents transferrin and hemin causes opposite effects on the TRAP promoter activity. Two regulatory GAGGC tandem repeat sequences (the hemin responsive elements, HRE) within the 5'-flanking region of the human TRAP gene were identified. Studies with specific HRE-deletion constructs of the human TRAP 5'-flanking region upstream of the luciferase reporter gene document the functionality of these HRE-sequences which are apparently responsible for mediating transcriptional inhibition upon exposure to hemin. In addition to the previously published functional characterization of the murine TRAP HRE motifs, these results provide the first description of a new iron/hemin-responsive transcriptional regulation in the human TRAP gene.

Topics: Acid Phosphatase; Cell Line; Gene Expression Regulation, Enzymologic; Hemin; Humans; Promoter Regions, Genetic; Tartrates; Transcription, Genetic

In free-living Amoeba proteus (strain B), acid phosphatase (AcP) was examined by disc-electrophoresis in polyacrylamide gel. The tartrate-sensitive amebian AcP was greatly inhibited by dithiothreitol and Cu2+, and only partly inhibited by sodium orthovanadate, ammonium molybdate, EDTA, disodium salt and Mg2+, Ca2+, Zn2+ and Mn2+. On the contrary, it appeared to be resistant to sulfhydryl reagents--4(hydroxymercury) benzoic acid, sodium salt and N-ethylmaleimide. Unlike the tartrate-sensitive enzyme, the tartrate-resistant AcP was greatly inhibited by EDTA and partly inhibited by dithiothreitol, Mg2+ and Cu2+ (Mn2+ > Cu2+), being activated by orthovanadate, molybdate, sulfhydryl reagents, Mg2+, Ca2+ and Zn2+. Both tartrate-sensitive and tartrate-resistant AcPs lack apparently free SH-groups necessary for their catalytic activities. Using 2-naphthyl phosphate as a substrate at pH 4.5, six AcP electromorphs were revealed in cytosol and sediment, four of these being most frequently localized in the former, and two in the latter. Two other AcP electromorphs were confined to the sediment only. Depending on the quantity of sedimented amoebae making a homogenate (0.5 or 2.0 cm3), that was added to Percoll solution, the lysosomal AcP fraction in polyacrylamide gel was represented by one or two tartrate-sensitive electromorphs. Therefore, tartrate-resistant AcP in A. proteus may be a lysosomal enzyme, while tartrate-resistant AcP may correspond to serine/threonine protein phosphatase.

Topics: Acid Phosphatase; Amoeba; Animals; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Inhibitors; Substrate Specificity; Tartrates

A major component of innate immune responses relies on monocytes and macrophages, virus infection of which will pose a particular problem for immunological defense. Consequently, the monocytic cell differentiation pathway was analyzed in terms of cellular modulations therein and their relation to monocytotropic virus infection. Differentiation was characterized by down-regulation of CD14, MHC Ags, the monocytic SWC1 marker, and p53; concomitant up-regulation of the SWC9 macrophage marker, a putative porcine CD80 (detected with anti-human CD80 Ab), and acid phosphatase secretion were also characteristic. Elevated phagocytic and endocytic activities as well as endosomal/lysosomal acidification were identified as being important to the macrophage. In contrast, monocytes possessed high accessory activity. This was multifactorial, concomitantly requiring 1) high MHC Ag expression; 2) enzyme activity of esterase, peroxidase, myeloperoxidase, and 5' nucleotidase in preference to glucosidase, galactosidase, and glucuronidase; and 3) elevated capacity for spontaneous IL-1 production. Only with all parameters was efficient stimulation of Ag-specific lymphocytes possible. These results point to a continuous process during differentiation, involving inter-related characteristics linking the more accessory monocyte to the scavenger macrophage, both in vitro and in vivo. Of particular interest was how these characteristics related to monocytotropic virus infection, and how a particular virus could show a clear preference for the differentiating macrophages. Such results not only further our understanding of porcine immunology, but also provide evidence and a potential model for the determination and characterization of monocytotropic virus-host cell interactions.

Topics: Acid Phosphatase; African Swine Fever; African Swine Fever Virus; Animals; Antigen-Presenting Cells; Antigens, Surface; Cell Differentiation; Cells, Cultured; Disease Susceptibility; Endocytosis; Hydrogen-Ion Concentration; Interleukin-1; Intracellular Fluid; Macrophages; Monocytes; Phagocytosis; Swine; Tartrates

This study was carried out to evaluate the clinical validity and usefulness of serum tartrate-resistant fluoride-sensitive acid phosphatase (TrFsACP) activity using 2,6-dichloro-4-acetylphenyl phosphate as substrate at pH 6.2 in metabolic bone diseases. The mean Z-scores of TrFsACP activity in patients on hemodialysis were higher than in healthy subjects (male: 2.04+/-1.98, n = 49, P < .05; female: 1.49+/-2.43, n = 39, P < .05) and increased with duration of hemodialysis (r = .516, P < .01). Bone alkaline phosphatase also was found to be significantly higher in hemodialysis patients (male: 0.93+/-1.49, P < .05; female: 1.66+/-2.42, P < .05) compared with normal subjects: but had lower correlation with duration of hemodialysis than TrFsACP (r = .277, P < .05). Ulcerative colitis (1.37+/-2.21, n = 15) in males showed a significantly higher Z-score of TrFsACP compared with control subjects (P < .05). The relationship of TrFsACP activity and ultrasound findings (stiffness; speed of sound [SOS]; broadband ultra sound attenuation [BUA]) in healthy women aged 30-75 years (n = 95) were inversely and significantly correlated with stiffness (r = -.465, P < .01 ), SOS (r = -.484, P < .01), and BUA (r = -.366, P < .01), but were age dependent. TrFsACP activity significantly correlated with stiffness (r = -.521, P < .05) and SOS (r = -.527, P < .05) only in the age group of 46-55 years. BUA (r = -.313, P > .05) did not correlate significantly in any subject in the present study. We conclude that serum TrFsACP activity is useful in the diagnosis and monitoring of bone turnover.

Topics: Acid Phosphatase; Adult; Aged; Arthritis, Rheumatoid; Biomarkers; Bone and Bones; Bone Remodeling; Case-Control Studies; Colitis, Ulcerative; Female; Fluorides; Humans; Male; Middle Aged; Osteogenesis Imperfecta; Osteoporosis; Predictive Value of Tests; Reference Values; Renal Dialysis; Tartrates; Ultrasonography

The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2 mM sodium fluoride, whereas for osteoclasts 50-100 mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralizing zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Bone and Bones; Bone Development; Bone Matrix; Calcification, Physiologic; Cartilage; Chondrocytes; Fixatives; Formaldehyde; Growth Plate; Isoenzymes; Lysine; Periodic Acid; Polymers; Rabbits; Tartrate-Resistant Acid Phosphatase; Tartrates; Tissue Fixation

Hemin inhibits transcription of the tartrate resistant acid phosphatase (TRAP) gene. Using deletion mutagenesis of the mouse TRAP 5'-flanking region, we previously identified a 27-bp DNA segment containing a central GAGGC tandem repeat sequence (the hemin response element [HRE]), which bound nuclear proteins (hemin response element binding proteins [HREBPs]) from hemin-treated cells and appeared to be responsible for mediating transcriptional inhibition in response to hemin. We now have used affinity binding to HRE-derivatized beads to identify four HREBP components with apparent molecular masses of 133-, 90-, 80-, and 37-kD, respectively. The 80- and 90-kD components correspond to the p70 and p80/86 subunits of Ku antigen (KuAg) as documented by partial amino acid microsequencing of tryptic digests and immunologic reactivity. Based on reactivity of the HREBP gel shift band with antibodies to the redox factor protein (ref1) in shift Western experiments, it is shown that the 37-kD component represents ref1. The 133-kD component appeared to be a unique protein. KuAg participation in HREBP complexes was specific as it was present in HREBPs bound to HRE microcircles. Results of depletion/reconstitution experiments suggested that KuAg does not bind alone or directly to HRE DNA, but does so only in conjunction with the 133- and/or 37-kD proteins. We conclude that HREBP is a heterogeneous complex composed of KuAg, ref1, and a unique 133-kD protein. We speculate that the role of heme may be to promote interactions among these components, thereby facilitating HRE binding and downregulation of hemin responsive genes.

Topics: Acid Phosphatase; Antigens, Nuclear; Base Sequence; Binding Sites; Cell Line; Cross-Linking Reagents; DNA; DNA Helicases; DNA-Binding Proteins; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Hemin; Humans; Ku Autoantigen; Molecular Weight; Nuclear Proteins; Repetitive Sequences, Nucleic Acid; Tartrates; Transcription, Genetic; Ultraviolet Rays

We describe an improved method for the kinetic measurement of tartrate-resistant acid phosphatase (TrACP; EC 3.1.3.2) activity in serum. Of the TrACP derived from erythrocytes, platelets, and macrophages (osteoclasts and others), that from the first two sources is also resistant to fluoride, whereas skeletal TrACP is sensitive to fluoride. Thus, osteoclast-derived TrACP can be measured specifically by exploiting its sensitivity to fluoride. We measured the activity of tartrate-resistant and fluoride-sensitive acid phosphatase (TrFsACP) by using 2,6-dichloro-4-acetylphenyl phosphate as substrate at pH 6.2. The activity of TrFsACP in serum was increased by adding hexadimethrine bromide (Polybrene) to the reaction mixture. This method was not influenced by hemolysis with hemoglobin concentrations as great as 0.9 g/L. The mean +/- SD values of TrFsACP activity by this method were 20.4 +/- 2.8 and 16.4 +/- 2.3 U/L for young (ages 20-29 years) men (n = 34) and women (n = 50), respectively. The highest mean TrFsACP activity was found among children younger than 15 years, followed by that in elderly subjects (older than 60).

Topics: Acid Phosphatase; Adult; Animals; Blood Platelets; Cattle; Enzyme Inhibitors; Erythrocytes; Female; Hemolysis; Heparin; Heparin Antagonists; Hexadimethrine Bromide; Humans; Isoenzymes; Kinetics; Male; Organophosphates; Osteoclasts; Sodium Fluoride; Tartrate-Resistant Acid Phosphatase; Tartrates

LY-117018 HCl is a recently developed, selective estrogen receptor modulator and an analog of raloxifene. Its mode of action on the skeleton is similar to that of estrogens, although it does not have the same side effects. The aim of the current study was to compare the effects produced by the administration of 17alpha-ethinyl estradiol (0.1 mg/kg/day) and LY-117018 HCl (1 mg/kg/day) on bone remodeling, bone mineral density, and body and uterus weight in sham-operated and oophorectomized rats (experimental model of postmenopausal osteoporosis).. Twelve-week-old female Wistar rats were used. Treatment was given for 3 months after oophorectomy or sham operation. Bone mineral density was determined in the lumbar spine (L2, L3, and L4) and in the left femur with use of a Hologic QDR 1000 (S/N 277) densitometer. Bone remodeling was estimated with use of the formation markers osteocalcin (bone gla protein) and alkaline phosphatase and the resorption markers type I collagen carboxyterminal telopeptide and tartrate-resistant acid phosphatase.. The newly developed derivative of raloxifene, LY-117018 HCl, offsets the reduction in bone mineral density and the accompanying increase in bone remodeling markers observed in oophorectomized rats compared with control animals. 17Alpha-ethinyl estradiol also prevents the loss of bone mass attributed to oophorectomy, but this is accompanied by an increase in body mass and a greater increase in uterus weight than that observed after treatment with LY-117018 HCl.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Body Weight; Bone Density; Bone Remodeling; Calcium; Collagen; Collagen Type I; Creatinine; Estrogen Antagonists; Ethinyl Estradiol; Female; Organ Size; Osteocalcin; Ovariectomy; Peptides; Pyrrolidines; Rats; Rats, Wistar; Tartrates; Thiophenes; Uterus

The pleiotropic cytokine interleukin-11 (IL-11) stimulates osteoclast formation in vitro, but it is not known whether it influences other steps in the bone-resorptive cascade. Using a variety of in vitro model systems for studying bone resorption we have investigated the effects of IL-11 on 1) osteoclast formation, fusion, migration, and activity; and 2) osteoblast-mediated osteoid degradation. The involvement of matrix metalloproteinases (MMPs) and products of arachidonic acid metabolism in IL-11-mediated resorption were also assessed. We first examined the bone-resorptive effects of IL-11 by assessing 45Ca release from neonatal mouse calvarial bones. IL-11 dose-dependently stimulated bone resorption with an EC50 of 10(-10) M. The kinetics of IL-11-mediated 45Ca release demonstrated that it was without effect for the first 48 h of culture, but by 96 h, it stimulated 45Ca release to the same level as that produced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (a hormone that stimulates osteoclast formation and activity). IL-11 also produced a dose-dependent increase in osteoblast-mediated type I collagen degradation with a maximum of 58.0 +/- 6.2% at 5 x 10(-9) M; this effect of IL-11 was less than that produced by 1,25-(OH)2D3 (76.5 +/- 7.1%) and was prevented by an inhibitor of MMPs, but not those blocking arachidonic acid metabolism. We then tested the effects of IL-11 on isolated mouse osteoclasts cultured on ivory slices in the presence and absence of primary mouse osteoblasts. IL-11 had no effect on isolated osteoclast activity even in coculture with primary osteoblasts. We then examined the effects of IL-11 on the formation of osteoclast-like multinucleate cells in mouse bone marrow cultures and the resorptive activity of such cultures using ivory as a substrate. IL-11 dose-dependently increased 1) the number of tartrate-resistant acid phosphatase-positive osteoclast-like multinucleate cells and 2) the surface area of lacunar resorption, although the effects were less than that of 1,25-(OH)2D3. The effect of IL-11 on bone marrow lacunar resorption was prevented by a combination of inhibitors of 5-lipoxygenase and cyclooxygenase. In 17-day-old metatarsal bones, IL-11 prevented the migration of (pre)osteoclasts to future resorption sites, whereas their fusion was unaffected. These results provide strong evidence that IL-11 stimulates bone resorption by enhancing osteoclast formation and osteoblast-mediated osteoid degradation rather than stimulating ost

Topics: Acid Phosphatase; Animals; Animals, Newborn; Arachidonic Acid; Bone and Bones; Bone Marrow Cells; Bone Resorption; Calcium Radioisotopes; Cell Fusion; Cell Movement; Cells, Cultured; Collagen; Drug Resistance; Humans; Interleukin-11; Metalloendopeptidases; Mice; Osteoblasts; Osteoclasts; Tartrates

Stromal cells are required for in vitro osteoclast differentiation and maturation. The murine bone marrow stromally derived BMS2 cell line exhibits adipocytic and osteoblastic features as well as the ability to support lymphopoiesis and myelopoiesis. This work examined the ability of the BMS2 cell in either the preadipocyte or adipocyte state to support the formation of osteoclast-like cells. BMS2 cells can be induced to undergo adipogenic differentiation in response to treatment with glucocorticoids or thiazolidinedione compounds. Primary bone marrow cells, enriched for hematopoietic progenitors and depleted of their adherent stromal and macrophage populations, were stimulated with vitamin D3 (vitamin D; 10(-8) M) to undergo osteoclast differentiation and maturation when cocultured with BMS2 cells. In both preadipocyte and adipocyte-enriched BMS2 stromal layers, comparable numbers of tartrate-resistant acid phosphatase-positive osteoclast-like cells, characterized by their response to salmon calcitonin with an increase in cAMP and formation of resorption pits on bovine bone slices, were formed. The gene expression and protein levels of macrophage colony-stimulating factor produced by preadipocyte and adipocyte-rich BMS2 layers were comparable. However, adipocyte-rich stromal layers supported osteoclast-like cell formation longer in culture than preadipocytes, independent of the agent used to induce adipocyte differentiation. These studies demonstrate for the first time that fully differentiated adipocyte stromal cells can support osteoclast-like cell formation and function in vitro.

Topics: Acid Phosphatase; Adipocytes; Animals; Bone Marrow Cells; Calcitonin; Cell Differentiation; Cell Line; Cholecalciferol; Coculture Techniques; Flow Cytometry; Hematopoietic Stem Cells; Macrophage Colony-Stimulating Factor; Mice; Osteoclasts; Stromal Cells; Tartrates; Thiazoles; Thiazolidinediones

Human prostatic acid phosphatase (PAcP) is a prostate epithelium-specific differentiation antigen. In prostate carcinomas, the cellular PAcP is decreased. We investigated its functional role in these cells. Several lines of evidence support the hypothesis that cellular PAcP functions as a neutral protein-tyrosine phosphatase and is involved in regulating prostate cell growth. In this study, we identify its in vivo substrate. Our results demonstrated that, in different human prostate cancer cell lines, the phosphotyrosine (Tyr(P)) level of a 185-kDa phosphoprotein (pp185) inversely correlates with the cellular activity of PAcP. On SDS-PAGE, this pp185 co-migrates with the c-ErbB-2 oncoprotein. Immunodepletion experiments revealed that c-ErbB-2 protein is the major pp185 in cells. Results from subclones of LNCaP cells indicated the lower the cellular PAcP activity, the higher the Tyr(P) levels of c-ErbB-2. This inverse correlation was further observed in PAcP cDNA-transfected cells. In clone 33 LNCaP cells, L-(+)-tartrate suppresses the cellular PAcP activity and causes an elevated Tyr(P) level of c-ErbB-2 protein. Epidermal growth factor stimulates the proliferation of LNCaP cells, which concurs with a decreased cellular PAcP activity as well as an increased Tyr(P) level of c-ErbB-2. Biochemically, PAcP dephosphorylates c-ErbB-2 at pH 7.0. The results thus suggest that cellular PAcP down-regulates prostate cell growth by dephosphorylating Tyr(P) on c-ErbB-2 oncoprotein in those cells.

Topics: Acid Phosphatase; Humans; Male; Molecular Weight; Phosphorylation; Prostate; Prostatic Neoplasms; Receptor, ErbB-2; Tartrates; Tumor Cells, Cultured; Tyrosine

It has been suggested that acid phosphatase activity is present in newly formed bone matrix at sites of endochondral ossification in rabbit fracture calluses. Because acid phosphatases are usually found intracellularly, it was decided to test this possibility more rigorously. Tissue from 10- and 14-day healing rabbit fractures was subjected to a series of critical tests for acid phosphatases with a pH optimum of 5.0. Fluoride, tartrate and molybdate were used as potential inhibitors of acid phosphatase activity. The effects of several counterstaining protocols were also investigated. A fluoride- and tartrate-resistant acid phosphatase is located in osteoclasts and mononuclear phagocytes. Diffuse staining of the bone matrix is seen, but it is dependent upon the length of incubation in the substrate medium and the distance from the acid phosphatase-reacting cells. It is concluded that the coloration of the bone matrix is probably caused by diffusion of the dye and reaction product and is, therefore, artifactual.

Topics: Acid Phosphatase; Animals; Artifacts; Bone Matrix; Bony Callus; Chondrocytes; Fluorides; Fracture Healing; Molybdenum; Osteoblasts; Osteoclasts; Osteogenesis; Phagocytes; Rabbits; Staining and Labeling; Tartrates; Tibial Fractures

To evaluate the relationship between biochemical markers of bone turnover and bone scan indices of disease activity, as well as to analyze their variations based on skeletal involvement, in Paget's disease.. Serum samples were obtained from 51 patients with Paget's disease to determine the levels of total alkaline phosphatase (total AP), bone alkaline phosphatase (bone AP), propeptide carboxyterminal of type I procollagen (PICP), propeptide aminoterminal of type I procollagen (PINP), osteocalcin, tartrate-resistant acid phosphatase, and telopeptide carboxyterminal of type I collagen. Urine samples were analyzed for levels of hydroxyproline (HYP), pyridinoline (PYR), deoxypyridinoline (DPYR), C-terminal telopeptide of type I collagen (CTx), and N-terminal telopeptide of type I collagen (NTx). In addition, 2 semiquantitative scintigraphic indices, disease activity (AI) and disease extent (EI), were obtained. Pagetic skeletal locations were evaluated individually, with special attention to skull involvement.. All biochemical markers correlated with the AI and the EI. Serum PINP, bone AP, and total AP showed the highest proportions of increased values among the bone formation markers (94%, 82%, and 76%, respectively). Among the bone resorption markers, urinary NTx showed the highest proportion of increased values in patients with Paget's disease (96%), compared with PYR (69%), DPYR (71%), CTx (65%), and HYP (64%). In patients with mild disease activity, serum PINP was the marker with the highest proportion of increased values (71%). In contrast, serum PICP and urinary CTx were the most discriminative markers for skull involvement. Except for higher values for most of the biochemical markers of bone turnover in flat bones, no major differences in other skeletal locations were observed.. The determination of serum PINP as a marker of bone formation and urinary NTx as a marker of bone resorption provided the best biochemical profile to ascertain the extent and activity of Paget's disease. In patients with skull involvement, serum PICP and urinary CTx were shown to be the most discriminative markers.

Topics: Acid Phosphatase; Adult; Aged; Aged, 80 and over; Alkaline Phosphatase; Biomarkers; Bone and Bones; Bone Resorption; Collagen; Female; Humans; Male; Middle Aged; Osteitis Deformans; Osteocalcin; Radionuclide Imaging; Tartrates

Lysosomal acid phosphatase (LAP) is a tartrate-sensitive enzyme with ubiquitous expression. Neither the physiological substrates nor the functional significance is known. Mice with a deficiency of LAP generated by targeted disruption of the LAP gene are fertile and develop normally. Microscopic examination of various peripheral organs revealed progredient lysosomal storage in podocytes and tubular epithelial cells of the kidney, with regionally different ultrastructural appearance of the stored material. Within the central nervous system, lysosomal storage was detected to a regionally different extent in microglia, ependymal cells, and astroglia concomitant with the development of a progressive astrogliosis and microglial activation. Whereas behavioral and neuromotor analyses were unable to distinguish between control and deficient mice, approximately 7% of the deficient animals developed generalized seizures. From the age of 6 months onward, conspicuous alterations of bone structure became apparent, resulting in a kyphoscoliotic malformation of the lower thoracic vertebral column. We conclude from these findings that LAP has a unique function in only a subset of cells, where its deficiency causes the storage of a heterogeneously appearing material in lysosomes. The causal relationship of the enzyme defect to the clinical manifestations remains to be determined.

Topics: Acid Phosphatase; Animals; Antigens, CD; Bone and Bones; Cathepsin D; Central Nervous System Diseases; Fibroblasts; Kidney Diseases; Lysosomal Membrane Proteins; Lysosomal Storage Diseases; Lysosomes; Membrane Glycoproteins; Mice; Microglia; Phenotype; Seizures; Tartrates

This study was carried out in order to evaluate clinical usefulness of cross-linked N-telopeptides (NTx) of type I collagen determination, in patients with primary hyperparathyroidism. Twenty-six consecutive patients (6 males and 20 females, aged 56.3 +/- 15.0, SD, yrs) with primary hyperparathyroidism were studied in basal conditions and, ten of them, after surgical cure of the disease. Cross-linked collagen peptides were measured by enzyme-linked immunosorbent assay and conventional markers of bone turnover according to standard procedures. Bone densitometry at the lumbar spine and proximal femur was performed using dual-energy X-ray absorptiometry. Bone mineral density, was also assessed at the junction of the distal and middle third of the radius and at the ultradistal radius of the non-dominant arm by a dual photon densitometer. Mean urinary NTx values (194.2 +/- 121.9 pmoles bone collagen equivalents/mumoles creatinine) were significantly higher (p < 0.001) in respect to those found in normal subjects. The mean increase of Z score values of both serum tartrate resistant acid phosphatase activity (1.4 +/- 1.8) and the fasting hydroxyproline/creatinine ratio (1.45 +/- 2.0) was significantly lower (p < 0.02) in respect to that of NTx Z score values (3.3 +/- 3.3); the latter values were not significantly different than mean Z score values of serum osteocalcin (4.0 +/- 3.9), serum alkaline phosphatase activity (2.6 +/- 2.6) and urinary calcium/creatinine ratio (3.2 +/- 3.3). We found a significant inverse correlation between NTx values and both lumbar spine (p < 0.01) and ultradistal radius bone mineral density (p < 0.05); a modest inverse correlation was also observed between serum tartrate resistant acid phosphatase activity and lumbar spine bone mineral density (p < 0.04). Following successful adenoma removal, the percentage decrease of both NTx and hydroxyproline was similar in patients with increased bone turnover rate; major discrepancies were observed in patients with normal values of NTx, the telopeptide reduction being greater than that of hydroxyproline. Finally, in a hypercalcemic patient with metastatic parathyroid cancer, telopeptide excretion was shown to be more sensitive in respect to urinary hydroxyproline when evaluating the effects of antiresorptive therapy. Our results seem to indicate that amongst the markers with good sensitivity, NTx is the only one that is inversely related with bone mineral density at two different skeletal sites

Topics: Absorptiometry, Photon; Acid Phosphatase; Adenoma; Adult; Aged; Alkaline Phosphatase; Bone Density; Bone Resorption; Calcium; Collagen; Collagen Type I; Creatinine; Enzyme-Linked Immunosorbent Assay; Female; Humans; Hydroxyproline; Hyperparathyroidism; Male; Middle Aged; Osteocalcin; Parathyroid Neoplasms; Peptides; Tartrates

The crystal structure of the complex between rat-prostatic acid phosphatase (PAP) and L-(+)-tartrate (Lindqvist et al., J. Biol. Chem., 1993, 268, 20744-20746) contains the model of the ligand with incorrect chirality. We report here the correct model and discuss the relation between this model and the model of the inhibitory complexes between PAP and oxy-anions.

Topics: Acid Phosphatase; Animals; Binding Sites; Enzyme Inhibitors; Hydrogen Bonding; In Vitro Techniques; Ligands; Male; Models, Molecular; Prostate; Protein Conformation; Rats; Static Electricity; Stereoisomerism; Tartrates

Inflammatory reactions in rheumatoid arthritis (RA) often cause severe joint destruction. However, the mechanism of bone destruction is still a matter of controversy. To determine whether multinuclear cells found in the rheumatoid synovium can resorb bone, isolated synovial cells were assessed for tartrate-resistant acid phosphatase (TRAP) staining and the ability to resorb bone in a dentine resorption assay. TRAP-positive multinuclear cells were found in six out of 10 samples. These six samples showed resorption pit formation on dentine slices. The other four samples did not form resorption pits. The results of this study demonstrate that TRAP-positive multinuclear cells isolated from the rheumatoid synovium form resorption pits on dentine slices. Our results suggest that inflamed synovial cells in rheumatoid joints might participate in bone destruction.

Topics: Acid Phosphatase; Arthritis, Rheumatoid; Bone Resorption; Cells, Cultured; Dentin; Humans; Microscopy, Electron, Scanning; Synovial Membrane; Tartrates

Human prostatic acid phosphatase (PAcP), a prostate epithelium-specific differentiation antigen, was determined to exhibit the endogenous protein tyrosine phosphatase activity. We investigated the phosphoprotein(s) that might be dephosphorylated by PAcP in human prostate carcinoma cells. Several lines of evidence were presented to show that the tyrosine phosphorylation level of a 185 kDa phosphoprotein (pp185) is negatively correlated with the cellular activity of PAcP. (i) In DU145, PC-3 and high passaged LNCaP prostate carcinoma cells that have no or low PAcP expression, the phosphotyrosine (p-tyr) level of pp185 was higher than that in low passaged LNCaP cells that express an endogenous PAcP. (ii) In LNCaP cells grown in the presence of L(+)-tartrate, an inhibitor of PAcP, the tyrosine phosphorylation of pp185 was increased. (iii) Mediated by Lipofectin, a cationic liposome, the incorporation of purified PAcP protein into DU145 cells resulted in the decreased phosphorylation of pp185. Thus, the results taken collectively demonstrated that the p-tyr level of pp185 is inversely correlated with the cellular activity of PAcP and indicated that the pp185 may be a putative substrate of PAcP in prostate carcinoma cells.

Topics: Acid Phosphatase; Humans; Male; Phosphorylation; Prostatic Neoplasms; Tartrates; Tumor Cells, Cultured; Tyrosine

Mature osteoclasts specifically express the purple, band 5 isozyme (Acp 5) of tartrate-resistant acid phosphatase, a binuclear metalloenzyme that can generate reactive oxygen species. The function of Acp 5 was investigated by targeted disruption of the gene in mice. Animals homozygous for the null Acp 5 allele had progressive foreshortening and deformity of the long bones and axial skeleton but apparently normal tooth eruption and skull plate development, indicating a rôle for Acp 5 in endochondral ossification. Histomorphometry and mineralization density analysis of backscattered electron imaging revealed widened and disorganized epiphyseal growth plates with delayed mineralization of cartilage in 6- to 8-week-old mutant mice. The membrane bones of the skull showed increased density at all ages examined, indicating defective osteoclastic bone turnover. Increased mineralization density was observed in the long bones of older animals which showed modelling deformities at their extremities: heterozygotes and homozygous Acp 5 mutant mice had tissue that was more mineralized and occupied a greater proportion of the bone in all regions. Thus the findings reflect a mild osteopetrosis due to an intrinsic defect of osteoclastic modelling activity that was confirmed in the resorption pit assay in vitro. We conclude that this bifunctional metalloprotein of the osteoclast is required for normal mineralization of cartilage in developing bones; it also maintains integrity and turnover of the adult skeleton by a critical contribution to bone matrix resorption.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Development; Bone Resorption; Calcification, Physiologic; Gene Deletion; Mice; Mutation; Osteopetrosis; Phenotype; Tartrates

Acid phosphatase (ACP) was detected in whole-cell lysates, membrane-bound and water-soluble fractions of Cryptobia salmositica (pathogenic and nonpathogenic vaccine strains), Cryptobia bullocki, and Cryptobia catostomi using p-nitro-phenylphosphate as the substrate. High activities were in acidic pH (3.0-5.5) and the optimal pH was 5.0 Highest ACP activity was in the membrane-bound fraction. The pathogenic strain of C. salmositica had significantly higher total ACP activity than the vaccine strain and the other 2 species. However, the activity in the pathogenic C. salmositica decreased significantly with prolonged in vitro cultivation. The membrane-bound ACP of the pathogenic C. salmositica had highest resistance to the ACP inhibitor, sodium tartrate.

Topics: Acid Phosphatase; Animals; Cypriniformes; Fish Diseases; Hydrogen-Ion Concentration; Kinetoplastida; Oncorhynchus mykiss; Protozoan Infections; Protozoan Infections, Animal; Protozoan Vaccines; Serial Passage; Tartrates; Time Factors

We examined the response of different biochemical markers of bone resorption to bisphosphonate therapy (400 mg of etidronate daily for 6 months) in mild Paget disease (n = 14). Urinary markers included hydroxyproline (OHP), total (T) and free (F) pyridinolines (Pyds) determined by HPLC, immunoreactive FPyds, immunoreactive TPyds, and the N- and C-terminal telopeptides of type I collage (NTx, CL). Serum measurements included tartrate-resistant acid phosphatase (TRAcP) and the C-terminal telopeptide of type I collagen (ICTP). ICTP and TRAcP showed a minimal response to therapy (% change at 6 months, -13.1 +/- 6.8 and -6.7 +/- 3.4, respectively). The response was greatest for urinary telopeptides (NTx and CL; % change -75.7 +/- 7.5 and -73.4 +/- 8.9, respectively). The response was somewhat greater for TPyds than for FPyds. We conclude that: (a) ICTP and TRAcP are unreliable indicators of changes in bone turnover; (b) oligopeptide-bound Pyds and telopeptide fragments of type I collagen in urine show a somewhat greater response to therapy than do FPyds and may be more sensitive indicators of bone resorption; and (c) as yet no evidence suggests that these markers are substantially better predictors of the clinical response to therapy than serum total alkaline phosphatase or urinary OHP. There are several problems with the interpretation of these measurements in Paget disease, and the clinical utility of these measurements remains uncertain.

Topics: Acid Phosphatase; Aged; Aged, 80 and over; Amino Acids; Biomarkers; Bone Resorption; Chromatography, High Pressure Liquid; Collagen; Drug Resistance; Etidronic Acid; Female; Humans; Hydroxyproline; Male; Middle Aged; Osteitis Deformans; Peptide Fragments; Tartrates; Testosterone

To study the role of parasite protein kinase C (PKC) activity in the uptake of Leishmania amazonensis by mononuclear phagocytes we treated the parasites with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and/or sphingosine, before interaction assays. Promastigotes of Leishmania amazonensis were incubated with 20 ng/ml TPA and/or 50 ng/ml sphingosine before the interaction with murine peritoneal macrophages. The short treatment enhanced about 200% the parasite association with the host cells, whereas the sphingosine treatment reduced about 50% the promastigote binding, as did the prolonged TPA treatment. The binding of cells treated with both drugs was not significantly altered. Biochemical and cytochemical data indicate that the protein kinase C agonists TPA and sphingosine, respectively, increased and decreased acid phosphatase (AcP) activity. The addition of sodium tartrate, a secreted AcP inhibitor, suppressed the TPA enhancing effects, but did not affect the basal parasite binding observed in control cells. The supernatants of TPA-treated L. amazonensis promastigotes increased the parasite association by about the same extent as the TPA treatment, and this effect was also abolished by tartrate. Although TPA did not enhance the association of L. major, a species that does not secrete AcP, the supernatants of TPA-treated L. amazonensis increased it in a tartrate-sensitive manner. The results suggest that Leishmania amazonensis PKC activity may modulate its interaction with macrophages via secreted AcP.

Topics: Acid Phosphatase; Animals; Cell Membrane; Flagella; Host-Parasite Interactions; Leishmania mexicana; Macrophages, Peritoneal; Mice; Protein Kinase C; Sphingosine; Tartrates; Tetradecanoylphorbol Acetate

Topics: Acid Phosphatase; Biomarkers; Bone Resorption; Drug Resistance; Humans; Osteoblasts; Osteoclasts; Tartrates

A tartrate-resistant acid phosphatase (TRACP) was purified from cord plasma by use of cation-exchange chromatography (carboxymethyl Sepharose), gel-filtration chromatography (Sephacryl S-200), and preparative isoelectric focusing. After raising polyclonal antibodies to the purified TRACP in rabbit, we used the antiserum to develop an ELISA. The antiserum cross-reacted with an extract of bone, but not with extracts of spleen, erythrocytes, platelets, or osteoblasts or with prostatic acid phosphatase. With this ELISA we determined the concentration of serum TRACP in healthy men to be 197 (61-301) micrograms/L (median and range). Serum TRACP concentrations were significantly higher in children and postmenopausal women and in patients with chronic renal failure, hyperparathyroidism, or hyperthyroidism. Estrogen replacement therapy of postmenopausal women for 3-6.5 months decreased their serum TRACP concentration by 70%.

Topics: Acid Phosphatase; Adolescent; Adult; Aged; Child; Chromatography, Gel; Drug Resistance; Enzyme-Linked Immunosorbent Assay; Estrogen Replacement Therapy; Female; Fetal Blood; Humans; Hydrogen-Ion Concentration; Isoelectric Focusing; Isoenzymes; Male; Middle Aged; Postmenopause; Premenopause; Reference Values; Sensitivity and Specificity; Tartrate-Resistant Acid Phosphatase; Tartrates

Tartrate-resistant acid phosphatase (TRACP) and carbonic anhydrase II (CA II) are key enzymes responsible for osteoclastic bone resorption. In this study, we proposed that estrogen loss in postmenopausal osteoporosis may enhance gene expression of TRACP and CA II, and subsequently increase osteoclastic bone resorption. We have, therefore, used the ovariectomized rat model of postmenopausal bone loss to investigate changes at the gene transcriptional level in osteoclastic bone-resorbing enzymes in ovariectomized (OVX) rats, sham ovariectomized (S-OVX) rats, and estrogen-treated ovariectomized (E-OVX) rats. We have demonstrated for the first time that ovariectomy in rats enhances gene expression of TRACP, and CA II. The mRNA levels in OVX were approximately three- and four-fold higher, respectively, than those in S-OVX. Enhancement was observed 1 week after ovariectomy and transcripts remain high during the experimental period of 8 weeks. Administration of 17 beta-estradiol to OVX (E-OVX) reduced gene expression of these osteoclastic bone-resorbing enzymes 18 hours after injection. It appeared that the suppression of the osteoclastic bone-resorbing enzymes by 17 beta-estradiol was most effective during the first 1-2 weeks but the degree of suppression was reduced at 8 weeks after ovariectomy. In conclusion, our results suggest that estrogen prevents bone loss by reducing the mRNA levels of osteoclastic bone-resorbing enzymes in bone tissue.

Topics: Acid Phosphatase; Animals; Bone Resorption; Carbonic Anhydrases; Disease Models, Animal; Estradiol; Female; Gene Expression Regulation, Enzymologic; Humans; Osteoclasts; Osteoporosis, Postmenopausal; Ovariectomy; Rats; Rats, Sprague-Dawley; Tartrates

The generation of monoclonal antibodies (MAbs) specific for tartrate-resistant acid phosphatase (TRAP) may aid development of a serological immunoassay for this marker of bone resorption. The lack of MAbs to TRAP largely reflects the difficulty in obtaining sufficient antigen for in vivo immunization strategies. We have circumvented this problem by using in vitro immunization, requiring a small amount of TRAP isolated from osteoclastoma tumor. Two MAbs designated 312D and 310A were generated that exhibited weak anti-TRAP activity in enzyme-linked immunosorbent assay and immunocytochemistry. A polyclonal antibody to TRAP (Ab8023) was also raised in rabbit, using synthetic peptide. Ab8023 and MAbs 310A and 312D exhibited no activity against TRAP in dot-blotting experiments. Further characterization in enzyme-liked immunosorbent assay showed that Ab8023 was remarkably specific for TRAP whereas MAb 312D cross-reacted with another metalloenzyme, human prostatic acid phosphatase.

Topics: Acid Phosphatase; Animals; Antibodies, Monoclonal; Antibody Formation; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Humans; Immunohistochemistry; Isoenzymes; Male; Mice; Prostate; Rabbits; Tartrate-Resistant Acid Phosphatase; Tartrates

We analyzed c-fos mRNA expression by northern blotting analysis in chicken osteoclast precursors which spontaneously differentiate to multinucleated osteoclasts in 5-6 days. Osteoclast precursors as well as mature multinucleated osteoclasts showed constitutive expression of c-fos mRNA which is not found in osteoblasts. The c-fos expression was enhanced transiently by serum, dibutyryl cAMP (10(-4) M) and phorbol 12-myristate 13-acetate (TPA) (5 x 10(-7) M). To clarify the role of c-fos in osteoclast differentiation, c-fos DNA was transfected into osteoclast precursors. Greater than 2 fold increases in tartrate resistant acid phosphatase (TRAP) and bone resorptive activity were observed in the transfected cells compared to controls 3 days after transfection, suggesting that prolonged expression of c-fos caused enhanced osteoclast differentiation.

Topics: Acid Phosphatase; Animals; Bucladesine; Cell Differentiation; Cells, Cultured; DNA; Gene Expression; Genes, fos; Osteoclasts; RNA, Messenger; Stem Cells; Tartrates; Tetradecanoylphorbol Acetate; Transfection

Murine long-term bone marrow cultures (LTBMCs) were used to generate hematopoietic cells free from marrow stromal cells. These progenitor cells were treated with GM-CSF (5 U/ml) with or without rat bone osteocalcin or rat serum albumin in either alpha-MEM with 2% heat-inactivated horse serum alone (alpha) or supplemented with 10% L-cell-conditioned medium (as a source of M-CSF) (L10). Few substrate-attached cells survived in basal alpha medium, but when treated with L10 medium or GM-CSF, they survived and proliferated. Osteocalcin did not significantly affect survival or proliferation. Subcultures of cells treated with GM-CSF had large numbers of multinucleated cells, more than half of which were tartrate-resistant acid phosphatase-positive (TRAP). Osteocalcin further promoted the development of TRAP-positive multinucleated cells; a dose of 0.7 microgram/ml osteocalcin promoted osteoclastic differentiation by 60%. Using a novel microphotometric assay, we detected significantly more tartrate-resistant acid phosphatase activity in the osteocalcin plus GM-CSF group (75.6 +/- 14.2) than in GM-CSF alone (53.3 +/- 7.3). In the absence of M-CSF, GM-CSF stimulated tartrate-resistant acid phosphatase activity, but osteocalcin did not have an additional effect. These studies indicate that osteocalcin promotes osteoclastic differentiation of a stromal-free subpopulation of hematopoietic progenitors in the presence of GM-CSF and L-cell-conditioned medium. These results are consistent with the hypothesis that this bone-matrix constituent plays a role in bone resorption.

Topics: Acid Phosphatase; Animals; Bone Marrow Cells; Cell Count; Cell Differentiation; Cell Division; Cell Survival; Cells, Cultured; Culture Media, Conditioned; Granulocyte-Macrophage Colony-Stimulating Factor; Mice; Mice, Inbred C57BL; Osteocalcin; Osteoclasts; Stem Cells; Tartrates; Time Factors

Osteoclasts contain tartrate-resistant acid phosphatase (TRAP). To understand the role of osteoclasts in trabecular bone formation, the distal spongiosa of the femurs from 1, 2, 4, 8, 16, and 24 week-old mice were histochemically examined in methacrylate sections stained with an azo-dye method for TRAP. For quantitative evaluations, the surface density of trabecular bones, length of the spongy bones, numerical density of TRAP-positive cells (osteoclasts and mononuclear cells), proportions of the trabecular bone surface covered with osteoclasts, numerical density of osteoclasts on the trabecular bone surface, size of osteoclasts, and distribution of TRAP-positive mononuclear cells in the bone marrow were examined. In conclusion, the results indicate that 1) in the spongiosa, osteoclasts are distributed numerously at the ossification front along the epiphyseal cartilage, junction between the primary and secondary spongiosas, and diaphyseal end of trabecular bones for modeling of the spongiosa, 2) osteoclasts function independently from osteoblasts at the ossification front and the diaphyseal end of trabecular bones, 3) spongiosa changes the amount of trabecular bones through the bone formation-resorption equivalent stage, bone formation dominant stage, and bone resorption dominant stage, 4) TRAP-positive mononuclear cells, probable preosteoclasts, are distributed in the bone marrow, and 5) TRAP-positive mononuclear cells appear frequently in the marrow around osteoclasts, suggesting that osteoclast activity produces a differentiation or migration factor for preosteoclasts.

Topics: Acid Phosphatase; Aging; Animals; Bone Marrow; Bone Marrow Cells; Cell Division; Female; Femur; Leukocytes, Mononuclear; Mice; Mice, Inbred Strains; Osteoclasts; Tartrates

We have studied the levels of a new biochemical marker of bone resorption, carboxyterminal cross-linked telopeptide of type I collagen (ICTP), in 26 healthy control subjects, 15 patients with primary hyperparathyroidism (PHPT) and 17 patients with secondary hyperparathyroidism (secondary HPT). Levels of ICTP in PHPT and secondary HPT have been correlated with those of serum tartrate resistant acid phosphatase (TRAP), another biochemical marker of bone turnover, and with serum levels of intact parathyroid hormone (iPTH). The ICTP levels of the control group were 2.07 +/- 0.58 micrograms l-1, n = 26, range 1.3-3.2. They were independent of sex and age in the studied age range (30-62 years). The ICTP levels of PHPT patients were 3.5 +/- 3.5 micrograms l-1, mean +/- SD, range 0.5-12.2 micrograms l-1, significantly higher than those of control subjects (p < 0.05). We found a significant linear correlation between values of ICTP and iPTH levels (p < 0.01), between values of ICTP and serum activity of TRAP (p < 0.01) and between iPTH and TRAP levels (p < 0.01) in patients with PHPT. The ICTP levels in patients with secondary HPT were higher than those of patients with PHPT, 46 +/- 37 micrograms l-1, range 12-167 micrograms l-1 (p < 0.001) due to the impaired renal clearance of this peptide. We did not find a significant linear correlation between values of ICTP and iPTH levels in the serum of patients with secondary HPT, although we found a significant correlation between levels of ICTP and levels of TRAP, both biochemical markers of bone turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acid Phosphatase; Adult; Aged; Bone Resorption; Collagen; Collagen Type I; Female; Humans; Hyperparathyroidism; Male; Middle Aged; Parathyroid Hormone; Peptides; Tartrates

A carboxyl-terminal peptide sequence ("osteostatin") from parathyroid hormone related protein has been shown to have an inhibitory effect on osteoclastic bone resorption--an action opposite to its amino-terminal sequence. In this study, we proposed that inhibition of osteoclastic bone resorption by osteostatin was associated with reduction of tartrate resistant acid phosphatase (TRAcP) activity in osteoclasts. Our results have indicated that osteostatin reduced TRAcP activity in a dose dependent manner. This effect of osteostatin was both sensitive (half maximal effect approximately 5 x 10(-13) M) and potent (maximum inhibition approximately 50% of control). In the first 90 min of treatment, however, reduction of TRAcP activity was erratic but became persistent and progressive when the time course was extended. Moreover, throughout the experimental period the levels of TRAcP activity in the culture medium had fallen significantly. It appears that osteostatin has a biphasic effect on TRAcP activity, inhibiting its secretion and either suppressing its synthesis or increasing its degradation. In addition, osteostatin induced rapid cellular retraction of both human and rat cultured osteoclasts, which was morphologically distinct from that produced by calcitonin.

Topics: Acid Phosphatase; Amino Acid Sequence; Animals; Cells, Cultured; Isoenzymes; Molecular Sequence Data; Osteoclasts; Parathyroid Hormone-Related Protein; Peptides; Proteins; Rats; Rats, Wistar; Tartrate-Resistant Acid Phosphatase; Tartrates

Osteoclasts are multinucleated cells which form by fusion of circulating mononuclear hemopoietic precursors. The nature of these precursor cells and the roles bone stromal cells and hormonal factors play in their differentiation to osteoclasts are unknown. We cocultured adherent murine blood monocytes (nonspecific esterase and F4/80 positive; tartrate-resistant acid phosphatase negative) with osteoblastic and fibroblastic stromal cell lines in the presence of 2 x 10(-8) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Tartrate-resistant acid phosphatase and calcitonin (CT) receptor-positive osteoclastic cells, which formed numerous resorption pits in vitro, were noted after only 4 days in coculture with UMR106 osteoblast-like cells. Resorption was seen in cocultures to which as few as 100 peripheral blood mononuclear cells had been added. 1,25-(OH)2D3 and contact with live bone stromal cells were absolute requirements for monocyte differentiation into bone-resorbing cells. Both salmon CT (5 IU/ml) and prostaglandin E2 (10(-6) M) significantly inhibited bone resorption. Thus, a significant proportion of the peripheral blood mononuclear cells in the monocyte fraction are capable of differentiating into cells showing the cytochemical and functional characteristics of osteoclasts. The presence of specific hormonal [1,25-(OH)2D3] and bone stromal cell elements is necessary for this process to occur; the resultant resorption can be modulated by known inhibitors of bone resorption, CT and prostaglandin E2.

Topics: Acid Phosphatase; Animals; Bone Resorption; Calcitonin; Cell Differentiation; Culture Techniques; Dinoprostone; Humans; Kinetics; Monocytes; Osteoclasts; Rats; Receptors, Calcitonin; Tartrates; Tumor Cells, Cultured

Tartrate-resistant acid phosphatase (TRAP) became known as the characteristic, albeit not specific, marker enzyme for hairy cell leukemia (HCL). It is also expressed in other types of leukemic cells and in a variety of normal hematopoietic cells under both physiological and artificial conditions. The role of this distinctive enzyme is still unknown. TRAP hydrolyzes several chemical compounds, but its physiological substrate remains to be elucidated. Here, the insertion of a human TRAP cDNA into mammalian expression vector pSBC-2 yielded a construct that encoded enzymatically active TRAP in transfected baby hamster kidney cells (BHK-21). TRAP was expressed transiently, as shown by Northern blot analysis, polyacrylamide gel electrophoresis, isoelectric focusing and cytochemical staining. BHK-21 cells over-expressing the enzyme had a growth rate that was approximately 50% of that observed in control cells. A stable expression could not be achieved. Recent evidence suggested that TRAP might function as a protein tyrosine phosphatase. The effect of TRAP on protein tyrosine phosphorylation was examined by immunoblotting with an anti-phosphotyrosine monoclonal antibody (MoAb). The level of tyrosine phosphorylation was clearly lower in transfected BHK-21-TRAP cells than in the control cultures. Phorbol ester-mediated induction of protein tyrosine phosphorylation could overcome the status of reduced phosphorylation in BHK-21-TRAP cells. Furthermore, upon treatment with 12-O-tetra-decanoylphorphol-13-acetate tyrosine phosphorylation reached similar levels to stimulated control cells, indicating the existence of a functional endogenous phosphorylation network. Thus, TRAP might function as an antagonistic counterpart of cellular protein tyrosine kinases and could be involved in the control of cellular activation, proliferation, and differentiation.

Topics: Acid Phosphatase; Animals; Blotting, Northern; Cell Division; Cells, Cultured; Cricetinae; Electrophoresis, Polyacrylamide Gel; Gene Expression; Histocytochemistry; Isoelectric Focusing; Kidney; Phosphorylation; Pilot Projects; Protein Tyrosine Phosphatases; RNA; Tartrates; Transfection; Tyrosine

Topics: Acid Phosphatase; Humans; Male; Prostatic Hyperplasia; Prostatic Neoplasms; Tartrates

Previous reports have demonstrated that hemopoietic progenitor cells derived from mouse bone marrow can form osteoclast-like cells when cultured in the presence of stromal cells and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. We show here that in cocultures of mouse bone marrow cells and a clonal chondrogenic cell line (C5.18), a stimulation of the number of tartrate-resistant acid phosphatase-positive (TRAP+) colonies is seen with or without the addition of 1,25-(OH)2D3 to the cultures. A large proportion of the TRAP+ cells had calcitonin receptors. In addition resorption lacunae were observed on bone slices on which cocultures were maintained, thus these cells had the characteristics of osteoclasts. The number of osteoclast-containing colonies that formed in cocultures varied with the plating density of the C5.18 cells and the length of time the C5.18 cells were cultured before adding mouse bone marrow. These results suggested that osteoclast differentiation decreased with increasing cartilage differentiation. C5.18 cells treated with 1,25-(OH)2D3 before coculture stimulated TRAP+ osteoclast colony formation to a greater extent than untreated C5.18 cells, whereas C5.18 cells cultured in the presence of dexamethasone before coculture inhibited TRAP+ osteoclast colony formation relative to untreated C5.18 cells. Since 1,25-(OH)2D3 inhibits and dexamethasone stimulates cartilage differentiation in C5.18 cells, these results agree with the view that chondroprogenitor cells stimulate osteoclast colony formation, whereas cultures containing predominantly mature chondrocytes do not. Osteoclast-containing colonies were frequently associated with colonies of alkaline phosphatase-positive (AP+) cells. This raised the possibility that C5.18 cells stimulated osteoclast differentiation indirectly by increasing the numbers of AP+ stromal cells from the marrow population, which in turn could stimulate osteoclast differentiation from marrow hemopoietic progenitors. In cocultures in which the C5.18 cells were physically separated from the marrow cells, we also observed increased numbers of TRAP+ colonies growing in association with large colonies of AP+ cells, suggesting that C5.18 cells release a soluble factor that mediates these effects.

Topics: Acid Phosphatase; Animals; Bisbenzimidazole; Bone Marrow Cells; Calcitriol; Cartilage; Cell Count; Cell Differentiation; Cell Line; Chromatin; Dexamethasone; Mice; Osteoclasts; Spleen; Staining and Labeling; Tartrates

The efficiency of DNA transfection into mammalian cell cultures has been monitored using a variety of reporter assays. However, the common procedures are expensive, time-consuming and usually cannot identify the transfected cell population directly. In the present communication we describe a simple, inexpensive and efficient method to directly identify DNA transfection in mammalian cells using tartrate-resistant acid phosphatase (TRAP) gene expression. The method involves the transfection of a plasmid (pCT3), which contains TRAP cDNA driven by a CMV promoter, into mammalian cells. The cells can then be stained for TRAP activity, and the transfection efficiency can be determined by simply counting the positively transfected cells in a defined area with a microscope. This method permits screening of mammalian cells for transfection efficiency in multi-well plates. After waiting 30-40 minutes to allow the TRAP assay to saturate, wells can be scored in 1-2 minutes with little difficulty in detecting the transfected cells.

Topics: Acid Phosphatase; Animals; beta-Galactosidase; Calcium Phosphates; Cells, Cultured; Cloning, Molecular; Cytomegalovirus; DNA, Complementary; Gene Expression; Humans; Promoter Regions, Genetic; Staining and Labeling; Tartrates; Transfection

Dichloromethylene bisphosphonate (clodronate), an inhibitor of bone resorption, has been administered subcutaneously (1.25 mg/day, 3 consecutive days) to control male albino Wistar rats (n = 8). Variations in the levels of serum alkaline phosphatase (AP) and osteocalcin (BGP), biochemical markers of bone formation, and in urinary hydroxyproline (OH-Prol) and serum tartrate-resistant acid phosphatase (TRAP), markers of bone resorption, have been analyzed. A significant decrease was observed in OH-Prol/creatinine and TRAP. We found a positive correlation between the percentage of decrease, with respect to basal values, of TRAP and OH-Prol/creatinine (p < 0.05). These results suggest that TRAP could be a useful marker to evaluate the changes of bone resorption induced by bisphosphonates in the rat together with the classical marker OH-Prol. A significant decrease was observed in AP. BGP also decreased significantly, but the decrease was relatively small compared to the coefficient of variation of the method. We suggest the preferential use of AP determination, instead of BGP, in the study of the effects produced by bisphosphonates on bone formation in rats, if hepatic function is maintained.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Biomarkers; Bone Development; Bone Resorption; Clodronic Acid; Creatinine; Hydroxyproline; Male; Osteocalcin; Rats; Rats, Wistar; Tartrates

Little information is available on the molecular mechanisms controlling osteoclastic bone resorption. We used tartrate-resistant acid phosphatase (TRAP) to begin to investigate the regulation of bone resorption at the molecular level. TRAP is expressed at high levels in osteoclasts and may play an important role in the bone resorptive process. Therefore, we isolated the murine TRAP gene from a mouse spleen genomic library and characterized its promoter. A restriction map was generated for the 17 kb TRAP insert. A 2 kb SmaI fragment, containing the 5'-flanking region, was subcloned and the nucleotide sequence determined. Sequence analysis of the SmaI fragment revealed the presence of numerous candidate transcription factor binding sequences, including those for AP1 and H-APF-1. The H-APF-1 site matches the consensus sequence for the IL-6-regulated transcription factor. An intron was identified at -1 to -393 bp relative to the ATG. The presence of an intron was confirmed by PCR analysis of RNA isolated from murine osteoclasts. Primer extension analysis indicated the presence of a transcription initiation site at -552 bp from the ATG. The region from -1846 to 2bp relative to the ATG initiation codon drove the transient expression of a luciferase reporter gene when transfected into HRE H9 rabbit endometrial cells. PMA treatment of HRE H9 cells enhanced luciferase transcription approximately threefold. These data suggest that the TRAP promoter is complex and contains multiple regulatory elements. The availability of the TRAP promoter may also permit production of transgenic mice, which can be used to develop previously unavailable osteoclast cell lines.

Topics: Acid Phosphatase; Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; DNA Primers; Exons; Female; Genomic Library; Humans; Introns; Mice; Molecular Sequence Data; Osteoclasts; Polymerase Chain Reaction; Promoter Regions, Genetic; Restriction Mapping; Spleen; Tartrates; Transcription, Genetic

We previously reported that 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] tissue-specifically stimulated the production of the third component of complement (C3) in bone in vitro and in vivo. In the present study, we examined the possible roles of C3 in bone using bone marrow cultures with antibodies against C3 and C3 receptors (Mac 1, 8C12, and 7G6) and the purified mouse C3 protein. The C3 protein produced preferentially by stromal cells in response to 1 alpha,25-(OH)2D3 was distributed in macrophage-like mononuclear cells and small cells with few nuclei. Adding anti-C3 antibody together with 1 alpha,25-(OH)2D3 to bone marrow cultures greatly inhibited not only the appearance of tartrate-resistant acid phosphatase (TRAP)-positive mononuclear and multinucleated cells, but also the growth of macrophage-like mononuclear cells and stromal cells. The inhibitory effect of anti-C3 antibody on osteoclast-like cell formation was most prominent when it was added between days 2-4 of the 6-day culture period, which corresponded to the late proliferative phase and the early differentiation phase of osteoclast development. Adding anti-C3 receptor antibodies also inhibited osteoclast-like cell formation induced by 1 alpha,25-(OH)2D3. When C3 receptors were detected by the binding of C3-coated sheep red blood cells or immunostaining, the localization of C3 receptor-positive cells coincided exactly with that of C3 protein-positive cells. C3 receptors were expressed mainly in macrophage-like mononuclear cells, TRAP-positive mononuclear cells, and TRAP-positive small cells with few nuclei. TRAP-positive large cells with many nuclei were totally negative for C3 receptors. When macrophage-colony-stimulating factor (M-CSF), the purified C3 protein, and 1 alpha,25-(OH)2D3 were added to bone marrow methylcellulose cultures, separately or in combination, M-CSF-dependent colony formation was strikingly inhibited by 1 alpha,25-(OH)2D3, but the inhibition was prevented by simultaneously adding C3. These results provide additional evidence that osteoclast progenitors are indeed cells of the monocyte-macrophage lineage. It is likely that the C3 produced by stromal cells in response to 1 alpha,25-(OH)2D3 is somehow involved in osteoclast development by potentiating M-CSF-dependent proliferation of bone marrow cells and induction of osteoclast differentiation.

Topics: Acid Phosphatase; Animals; Bone Development; Bone Marrow Cells; Calcitriol; Cell Differentiation; Cell Division; Cells, Cultured; Complement C3; Macrophage Colony-Stimulating Factor; Macrophages; Mice; Monocytes; Osteoclasts; Receptors, Complement; Tartrates

In the last years, a parathyroid hormone (PTH)-related peptide (PTHrP) has been isolated from tumors associated with humoral hypercalcemia with malignancy (HHM). In the present work, we studied the effect of bovine PTH (bPTH)(1-34) and PTHrP(1-34) on tartrate-resistant acid phosphatase (TRAP), a marker of bone resorption, and alkaline phosphatase (AP) activities, and basal and vitamin D-stimulated osteocalcin (BGP) synthesis (markers of bone formation) in fetal rat calvaria cultures. After a 48-hour incubation period, both bPTH(1-34) and PTHrP(1-34) caused an increase in TRAP activity liberated in the medium with respect to control cultured calvaria. On the other hand, while after 2 or 4 h of incubation both bPTH(1-34) and PTHrP(1-34) caused a decrease in the AP activity liberated in the medium, after 48 h of incubation both peptides caused a significant increase in the AP liberated in the medium with respect to control cultures. With respect to BGP synthesis, both bPTH(1-34) and PTHrP(1-34) antagonized the 1,25-dihydroxyvitamin D3 stimulatory effect in calvaria cultures. We conclude that PTHrP(1-34) causes similar effects on bone, in organ cultures, to those caused by bPTH(1-34), namely an increase in both bone resorption and formation and a decrease in the vitamin D-stimulated BGP synthesis.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Bone and Bones; Calcitriol; Cells, Cultured; Osteocalcin; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Peptide Fragments; Proteins; Rats; Rats, Wistar; Tartrates; Time Factors

The crystal structure of recombinant rat prostatic acid phosphatase in complex with the inhibitor L(+)-tartrate was determined to 3-A resolution with protein crystallographic methods. The inhibitor binds at the carboxyl end of the parallel strands of the alpha/beta domain. One of the carboxyl groups of the tartrate molecule interacts with the conserved residues Arg-11, His-12, and Arg-15, which form part of the phosphate binding site. Furthermore, the C2 and C3 hydroxyl groups interact with His-257 and Arg-79. The second carboxyl group is close to Arg-79 but makes no direct hydrogen bonds to the protein. A sequence comparison between tartrate-sensitive and -resistant acid phosphatases suggests that these enzymes have different three-dimensional structures.

Topics: Acid Phosphatase; Amino Acid Sequence; Animals; Hydrogen Bonding; Molecular Sequence Data; Protein Conformation; Rats; Recombinant Proteins; Tartrates

Forty Japanese patients with hairy cell leukemia (HCL) were reviewed. Nine cases were diagnosed as typical HCL, and two cases had the features of HCL variant (prolymphocytic variant). The remaining 29 cases (72.5%) differed morphologically and hematologically from the other two groups in that they usually had a moderately high leukocyte count (average 27.9 x 10(3)/microliters), and abnormal cells showing a densely stained round nucleus and an inconspicuous nucleolus. Tartrate-resistant acid phosphatase reaction was weak, and their cells exhibited generally smooth or slightly irregular, cellular outlines in smears. The cells showed weak expression of surface immunoglobulin G (IgG) with kappa-chain predominance. CD25 antigen was not detected. Some of these findings resemble those of B-cell chronic lymphocytic leukemia, but the patients also had several important features of HCL. They had splenomegaly without significant lymphadenopathy. The abnormal cells were CD20+, CD11c+ and showed typical 'hairy morphology' under phase-contrast and scanning electron microscopy. Furthermore, spleen sections revealed diffuse infiltration by the abnormal cells in the red pulp. From these findings, we speculated that this group of patients constitute a distinct subtype of HCL which is commonly seen in Japan. We propose to term the disease as HCL Japanese variant.

Topics: Acid Phosphatase; Adult; Aged; Aged, 80 and over; Antigens, Surface; Drug Resistance; Female; Humans; Japan; Leukemia, Hairy Cell; Male; Microscopy, Electron, Scanning; Middle Aged; Tartrates

Previous studies have demonstrated that G-protein agonists induce quiescence (Q effect) or retraction (R effect) in isolated osteoclasts. We now report the functional effects of such agonists on osteoclastic bone resorption and enzyme release. Exposure of osteoclasts to tetrafluoro-aluminate anions (AlF4-), a universal G protein stimulator, resulted in a marked concentration-dependent inhibition of bone resorption. This was associated with a dramatic increase in the secretion of the osteoclast-specific enzyme, tartrate-resistant acid phosphatase (TRAP). Cholera toxin, a Gs stimulator and a selective Q effect agonist, similarly abolished bone resorption and enhanced TRAP secretion. In contrast, pertussis toxin, a Gi inhibitor and a selective R effect agonist, inhibited bone resorption significantly, but slightly reduced enzyme release. The results suggest an involvement of a Gs-like G protein in TRAP secretion from the osteoclast, possibly through a cyclic AMP-dependent mechanism.

Topics: Acid Phosphatase; Aluminum; Aluminum Compounds; Animals; Anions; Bone Resorption; Calcitonin; Cell Death; Cholera Toxin; Fluorides; Fluorine; GTP-Binding Proteins; Osteoclasts; Pertussis Toxin; Rats; Tartrates; Virulence Factors, Bordetella

Bryostatin 1 (Bryo1), a macrocyclic lactone and a protein kinase C activator, is extracted and purified from the marine bryozoan Bugula neritina. In this study we describe its effect on morphology, surface immunophenotype, acid phosphatase (AcP), tartrate-resistant acid phosphatase (TRAP), proliferation and cell cycle of non-Hodgkin's B-lymphoma cell lines representing four differentiation stages. Except for the WSU-BL, a high-grade SCNCL, all other cell lines showed obvious changes in their morphology when treated with 200 nM Bryo1. Phenotypically, a dramatic decrease of CD10 and induction of CD11c and BL7 on some cell lines consistent with further B-cell differentiation was seen. The lines in control cultures showed variable expression of AcP and TRAP. Following treatment with Bryo1, there was a general increase in AcP expression except in WSU-BL line. WSU-FSCCL and WSU-DLCL were TRAP-negative but became TRAP-positive when treated with Bryo1. Cell growth and cycle analysis during treatment of different cell lines revealed evidence of strong, moderate, or no growth inhibition by Bryo1 compared with control cultures. Our results indicate that Bryo1 shows differentiation effects on low-grade FSCCL, intermediate-grade FLCL and high-grade DLCL, and stimulatory or no effect on high-grade SCNCL. Since Bryo1 does not have tumor-promoting activity, it has a potential therapeutic role as a B-cell differentiating agent.

Topics: Acid Phosphatase; Antigens, CD; Antineoplastic Agents; Bryostatins; Cell Cycle; Cell Differentiation; Cell Division; Humans; Lactones; Lymphoma, B-Cell; Macrolides; Phenotype; Tartrates; Tumor Cells, Cultured

Tartrate-resistant acid phosphatase is a marker enzyme for osteoclasts, the multinucleated cell responsible for bone resorption. Interspecific somatic whole cell hybrids and karyotypically simple microcell hybrids were used to map the gene encoding tartrate-resistant acid phosphatase (Acp5) to mouse Chromosome 9. Acp5 is therefore a member of a syntenic family of genes that map to human chromosome 19p13.1-p13.3 and mouse Chromosome 9.

Topics: Acid Phosphatase; Animals; Base Sequence; Chromosome Mapping; Cricetinae; Cricetulus; DNA, Single-Stranded; Hybrid Cells; Mice; Molecular Sequence Data; Rats; Tartrates

Uteroferrin (Uf) is a purple acid phosphatase with a bi-iron center. It is the major secretory product of the porcine uterus under the influence of progesterone and supplies iron to the developing fetuses during pregnancy. Tartrate-resistant acid phosphatases (TRAP) are clearly similar to Uf in many of their properties but are generally located intracellularly in lysosomes. To determine whether Uf and intracellular TRAP are distinct gene products, cDNA for the TRAP from pig spleen were compared with Uf cDNA. Although no full-length cDNA for the former were isolated, a TRAP cDNA of 1.1 kilobases was identical in nucleotide sequence to a Uf cDNA (1.42 kilobases) in the region of overlap, which included the entire 3'-end of the transcript and most of the open reading frame. TRAP purified from porcine spleen also had an NH2-terminal amino acid sequence that corresponded to that of Uf purified from uterine secretions and was also similar in sequence to intracellular TRAP isolated from tissues of other species, including ones from human osteoclastomas and spleen. Finally, Southern hybridization analysis with two probes specific for exons 1 and 2 of the Uf gene strongly suggested the presence of only a single gene for acid phosphatases of this class in the pig. A similar analysis performed on human DNA with an exon-specific probe for human TRAP was also consistent with a single gene. It is concluded that the difference in trafficking between a secreted TRAP, such as Uf, and TRAP located in lysosomes is not the result of distinctive primary sequence of the polypeptides and that the variability within species ascribed to such enzymes is most likely the result of minor posttranslational changes.

Topics: Acid Phosphatase; Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; DNA; Exons; Female; Humans; Isoenzymes; Metalloproteins; Molecular Sequence Data; Polymerase Chain Reaction; Restriction Mapping; Sequence Homology, Amino Acid; Spleen; Swine; Tartrate-Resistant Acid Phosphatase; Tartrates; Uterus

The activity concentration and the specific activity (the ratio of enzyme activity to total serum protein) of the tartrate-inhibitable fraction of acid phosphatase [orthophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2; TIAP] were related to benign prostatic hypertrophy and to prostatic carcinoma. As expected, the TIAP activity concentrations assayed in the sera of patients with benign prostatic hypertrophy were within the range of those assayed in normal human sera. In contrast, the specific activities of TIAP determined in the sera of patients with benign prostatic hypertrophy were significantly higher than those determined in the control group. In the sera of prostatic carcinoma patients, both the TIAP activity concentrations and the TIAP specific activities differed significantly (F = 730) from the normal values.

Topics: Acid Phosphatase; Aged; Blood Proteins; Humans; Male; Middle Aged; Prostatic Hyperplasia; Prostatic Neoplasms; Reference Values; Tartrates

Topics: Acid Phosphatase; Antigens, CD; CD11 Antigens; CD5 Antigens; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Prolymphocytic; Tartrates

Osteoclasts express high levels of a vacuolar H(+)-adenosinetriphosphatase (H(+)-ATPase) on the ruffled membrane which they employ to dissolve bone mineral by acidifying their site of attachment on bone. The factors that control amplification of H(+)-ATPase during osteoclast differentiation are poorly understood. We examined the expression of vacuolar H(+)-ATPase in a cell culture system in which mouse spleen cells can be induced to differentiate into osteoclasts by coculture with a mouse bone marrow stromal cell line. We found that the coculture system produced active osteoclasts, identified as multinucleated cells with staining for tartrate-resistant acid phosphatase activity that formed genuine resorption pits in bone. These cells developed high levels of H(+)-ATPase expression in culture, and omission of dexamethasone or 1 alpha,25-dihydroxyvitamin D3 from the coculture system each partially suppressed the expression of H(+)-ATPase. The results demonstrate that the spleen and PA6 cell coculture system may be useful for investigating the factors that control the induction of H(+)-ATPase amplification that occurs during osteoclast differentiation.

Topics: Acid Phosphatase; Animals; Bone Resorption; Cell Differentiation; Cells, Cultured; Cytological Techniques; Dexamethasone; Dihydroxycholecalciferols; Drug Resistance; Genetic Markers; Mice; Microscopy, Electron, Scanning; Osteoclasts; Phenotype; Proton-Translocating ATPases; Spleen; Tartrates; Vacuoles

1. Chicken skeletal tartrate-sensitive (TsACP) and -resistant (TrACP) acid phosphatase isoenzymes could be separated from each other by carboxylmethyl-sepharose ion exchange chromatography. 2. Chicken skeletal TsACP showed a gradual time-dependent loss of sensitivity to tartrate inhibition when incubated at room temperature, but not at 4 degrees C. 3. The loss of sensitivity to tartrate inhibition was associated with an activation of the enzyme activity. 4. These changes were accompanied with a shift in the electrophoretic mobility of the enzyme activity from a large molecular sized form to a smaller molecular sized form that resembled the freshly prepared TrACP on the native acidic polyacrylamide electrophoresis gels, and on molecular sieve Superose-12 Fast Protein Liquid Chromatography. 5. Kinetic evaluations of the biochemical properties of the "converted" TsACP activity resembled the TrACP. 6. The apparent "conversion" was not unique to chicken TsACP, since similar "conversion" was observed with partially purified preparations of bovine bone matrix TsACP and of human osteoblastic TsACP. 7. Addition of several serine protease inhibitors did not prevent the "conversion". 8. These findings are consistent with the possibility that skeletal TsACPs are precursors of skeletal TrACPs.

Topics: Acid Phosphatase; Animals; Bone and Bones; Cattle; Cells, Cultured; Chick Embryo; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Humans; Isoenzymes; Kinetics; Substrate Specificity; Tartrate-Resistant Acid Phosphatase; Tartrates

The human nonerythrocytic acid phosphatases (AcP) are composed of seven distinct activity bands in nondenaturing polyacrylamide gel electrophoresis (PAGE) when stained using either 1-naphthyl phosphate or naphthol ASBI phosphate as substrate. They are numbered 0, 1, 2, 3, 3b, 4, and 5 according to their increasing mobility toward the cathode in acidic conditions. Of these, only the most cationic "band 5" is tartrate resistant (TRAcP). When naphthol ASBI phosphate is used as substrate, AcP activity can also be stained in situ. In the presence of tartrate, activity remains strong in the hairy cells (HC) of hairy cell leukemia (HCL). Thus, the TRAcP stain has remained a reliable marker for HC. To investigate the function of TRAcP in HC, we purified two isoforms of TRAcP from HCL spleen tissue and found them to have similar substrate specificities and inhibitor sensitivities. In this report, we describe in detail the methods for TRAcP purification and compare some of the structural properties of the two isoforms to reinforce the concept that human TRAcP is a heterogeneous group of related enzymes. Band 5 represented only 15-20% of the total TRAcP extracted from HCL spleen. The remaining 80% of TRAcP hydrolyzed p-nitrophenyl phosphate but not naphthol ASBI phosphate and was not detectable in acidic, nondenaturing PAGE gels. Band 5 was solubilized from tissue using 500 mmol/L NaCl after previous extraction with 0.5% (v/v) NP-40 removed most other AcP and TRAcP activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acid Phosphatase; Amino Acid Sequence; Biomarkers, Tumor; Colorimetry; Electrophoresis, Polyacrylamide Gel; Humans; Isoenzymes; Leukemia, Hairy Cell; Molecular Sequence Data; Molecular Weight; Species Specificity; Spleen; Tartrate-Resistant Acid Phosphatase; Tartrates

The present study provides a novel assay system to examine the differentiation of osteoclast progenitors on devitalized bone slices. We used the population of bone cells liberated enzymatically from 14-day-old mouse embryonal calvariae as a source of osteoclast progenitors. The analysis of differentiation of osteoclast progenitors into preosteoclasts and mature osteoclasts was assessed in terms of the formation of TRAP-positive cells and pits or resorption lacunae, respectively, on devitalized bone slices. Osteoclasts having bone-resorbing activity appeared when the calvarial cell population was cultured in the presence of 1 alpha,25-(OH)2D3 on devitalized bone slices. The resorbing activity increased in a 1 alpha,25-(OH)2D3 dose-related manner. However, calcitonin, a potent inhibitor of differentiation and activation of osteoclast lineage cells, reduced the area of the resorption lacunae in a dose-dependent fashion. The bone-resorbing cells on the bone slices expressed an obvious ruffled border and clear zone, structures specific to mature osteoclasts. These results suggest that osteoclast progenitors in the mouse calvarial population examined differentiated into mature osteoclasts in the presence of 1 alpha,25-(OH)2D3 on devitalized bone slices. Further, using this assay system we assessed the effect of some other osteotropic factors on the differentiation of osteoclast progenitors to mature osteoclasts. IL-1, IL-6, and PTH increased the formation of TRAP-positive cells and pits and the area of resorption lacunae in a dose-dependent fashion. However, prostaglandin E2 was unable to induce the formation of resorption lacunae, although a significant appearance of TRAP-positive cells was observed at a concentration of 200 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acid Phosphatase; Animals; Biological Assay; Bone and Bones; Bone Resorption; Calcitonin; Calcitriol; Cattle; Cell Differentiation; Cells, Cultured; In Vitro Techniques; Indomethacin; Mice; Mice, Inbred ICR; Microscopy, Electron; Osteoclasts; Skull; Stem Cells; Tartrates

Prolonged glucocorticoid excess is associated with bone loss. Among the contributory factors are glucocorticoids' suppression of bone formation and stimulation of bone resorption. In this study, the effects of glucocorticoids on bone resorption were evaluated in a rodent model. Subcutaneous implants of devitalized mineralized bone particles (BPs) elicit the recruitment of progenitor cells and their differentiation to osteoclasts which resorb the BPs. The effects of glucocorticoids on both the recruitment and the activity of cells induced by normal BPs were distinguished based upon when treatment was initiated. When treatment with hydrocortisone or dexamethasone was initiated at the time of BP implantation, the recruitment of bone-resorbing cells was impaired and a subsequent decrease in BP resorption was found. On the other hand, when treatment was initiated on day 7, glucocorticoids increased osteoclastic resorption and tartrate-resistant acid phosphatase activity. We also tested hydrocortisone's effect to stimulate the activity of cells associated with osteocalcin-deficient BPs. As previously reported, BPs deficient in osteocalcin were poorly resorbed as a result of decreased formation and activity of osteoclasts. Hydrocortisone had an even more pronounced effect in stimulating the low level resorption of the osteocalcin-deficient BP implants than of the normal BP implants. These findings show differential effects of glucocorticoids on two aspects of bone resorption: they inhibit the recruitment and/or differentiation of bone-resorbing cells, but they stimulate the activity of existing osteoclastic cells. The ability of glucocorticoids to increase resorption of normal bone and to overcome resistance to resorption of osteocalcin-deficient bone suggests an important regulatory effect of glucocorticoids in the activation of osteoclasts to increase bone resorption.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Resorption; Bone Transplantation; Cell Differentiation; Dexamethasone; Glucocorticoids; Hydrocortisone; Osteocalcin; Osteoclasts; Rats; Tartrates

1. Amino acid composition and immunological properties of the frog liver LMW AcPase forms: AcPase III and IV were examined. 2. AcPase III and IV show nearly identical amino acid composition and close immunological similarity. 3. These results indicate protein identity of both the enzyme forms and together with our previous data [Kubicz A., Szalewicz A. and Chrambach A., Int. J. Biochem. 23, 413-419 (1991)] demonstrate that generation of AcPase III and IV is a modification of the same enzyme protein by glycosylation processes. 4. Differences in immunoreactivity between AcPase III and IV were observed and discussed to be due to their altered conformations.

Topics: Acid Phosphatase; Amino Acids; Animals; Glycosylation; Immunodiffusion; Liver; Molecular Weight; Protein Processing, Post-Translational; Rana esculenta; Tartrates

Topics: Acid Phosphatase; Bone Neoplasms; Carbonic Anhydrases; Giant Cells; Humans; Isoenzymes; Tartrates

Tartrate-resistant acid adenosine triphosphatase activity at pH 6.5, using a lead-salt method, was localized at light and electron microscopic levels in cartilage and bone matrices, osteoclasts, and chondroclasts. Cartilage matrix staining occurred after vascular invasion of the growth plate. In osteoclasts, activity was present in lysosomes, extracellular ruffled border channels, and the underlying cartilage and bone matrices. Staining artifacts occurred at lower pH levels (pH 5.4, 5.0). Adenosine diphosphate, p-nitrophenylphosphate, thiamine pyrophosphate, and alpha-naphthylphosphate also acted as substrates; but no activity was observed when adenosine monophosphate, adenylate-(beta, gamma-methylene) diphosphate, and beta-glycerophosphate were used. The activity was inhibited by NaF, dithionite, and a high concentration of p-chloromercuribenzoic acid, and activated by simultaneous addition of FeCl2 and ascorbic acid, as has been shown in biochemical studies. These histochemical results support the view that the adenosine triphosphate hydrolyzing activity at pH 6.5 is due to tartrate-resistant acid phosphatase (TRAP). There were some differences in ultrastructural localization between TRAP and tartrate-sensitive acid phosphatase (TSAP) activities in osteoclasts: TSAP activity was more intense in lysosomes and Golgi complexes and TRAP was stronger in the cartilage and bone matrices. It is suggested, therefore, that most of TRAP is in an inactive form in cells and is activated when secreted.

Topics: Acid Phosphatase; Adenosine Triphosphatases; Animals; Bone Matrix; Cartilage; Chickens; Histological Techniques; Osteoblasts; Osteoclasts; Phosphoric Monoester Hydrolases; Tartrates; Tibia

Five main acid phosphatase (AcP) zones have been recognized and studied by polyacrylamide-gel electrophoresis. Band 5 represents the only tartrate-resistant form and is present in bone osteoclasts and in human alveolar macrophages (AMs). This study was carried out to quantify the presence of total and tartrate-resistant AcP (TrAcP) in AMs from bronchoalveolar lavage (BAL) of 11 patients with first stage sarcoidosis and in 13 nonsmokers and 16 smokers serving as control healthy subjects. The AMs from smokers showed an increase in total AcP activity (115.9 +/- 77.8 mU/10(6)); on the contrary, macrophages of patients with sarcoidosis revealed a consistent decrease in total AcP (27.8 +/- 7.0 mU/10(6)) and particularly the TrAcP subtype (14.8 +/- 3.7 mU/10(6)) in comparison with control nonsmokers (AcP = 42.2 +/- 18.9 mU/10(6) [p = NS]; TrAcP = 35.1 +/- 15.1 mU/10(6) [p less than 0.005]). The decrease in TrAcP activity was inversely correlated with the lymphocyte number (r = -0.75; p less than 0.01), lymphocyte percentage (r = -0.62; p less than 0.05), and CD4/CD8 ratio (r = -0.61; p less than 0.05). After six months of follow-up, the cytologic BAL picture returned completely to normal in five patients with full spontaneous regression of sarcoidosis; and also at the same time, normal values of TrAcP activity were restored. Since TrAcP activity can be easily detected, its possible use, along with the lymphocyte count and CD4/CD8 ratio, as a prognostic indicator of the clinical course of sarcoidosis deserves further investigation.

Topics: Acid Phosphatase; Adult; Bronchoalveolar Lavage Fluid; CD4-Positive T-Lymphocytes; Female; Histocytochemistry; Humans; Leukocyte Count; Lung Diseases; Macrophages; Male; Pulmonary Alveoli; Regression Analysis; Sarcoidosis; Smoking; T-Lymphocytes, Regulatory; Tartrates

Alkaline phosphatase (AlP) and tartrate-resistant acid phosphatase (TRAP) activities have been studied comparatively in needle biopsies of the iliac crest of four cases of secondary hyperparathyroidism (renal osteodystrophy). AlP activity was associated with the plasma membrane of osteoblasts and their processes, of reticular cells of bone marrow and of young osteocytes of osteoid borders and woven bone. Moreover, it was detected in the fibroblast-like cells of the endosteal "fibrosis". These cells were orderly in arrangement and were parallel to the endosteal surfaces near zones of bone formation. They were disorderly near zones of bone resorption. A strong TRAP-positive reaction was present in osteoclasts and mononuclear cells of endosteal "fibrosis" and in osteocytes located near active osteoclasts and in woven bone. These results suggest that the so-called fibrosis of hyperparathyroidism, rather than representing reparative, inert tissue, consists of osteoblast-like cells, probably precursors of osteoblasts derived by parathormone-stimulated proliferation of AlP-positive stromal cells of bone marrow, and of TRAP-positive, mononuclear cells, probably preosteoclasts. Moreover, they show that TRAP activity can be present in osteocytes, probably under stimulation by the same factors which stimulate osteoclast activity. The histochemical demonstration of AlP and TRAP facilitates the morphological diagnosis of metabolic bone disease and may improve knowledge of bone physiopathology.

Topics: Acid Phosphatase; Alkaline Phosphatase; Biopsy; Bone and Bones; Cold Temperature; Drug Resistance; Histocytochemistry; Histological Techniques; Humans; Hyperparathyroidism; Methacrylates; Tartrates; Tissue Embedding

Osteoclasts collected from the long bones of mice were cultured on dentin slices. To identify osteoclasts, the tartrate-resistant acid phosphatase (TRACPase) activity of cultured cells was histochemically examined by the azo dye method. The TRACPase-positive cells could be distinguished from other cells by light microscopy. The cells were sectioned by alternating semithin and ultrathin sections to observe their ultrastructure and three-dimensional structure. TRACPase activity was detected both in multi-nucleated osteoclasts and in mononuclear cells. Most of the mononuclear TRACPase-positive cells had features similar to preosteoclasts. A mononuclear TRACPase-positive cell was a ruffled border and clear zone was reconstructed three-dimensionally by NIKON COSMOZONE 2SA. The reconstruction showed that this cell possessed a large clear zone and small ruffled border. Under the ruffled border, no lacuna was apparent; but there was disruption of the dentin surface. The results suggest that this cell was a mononuclear osteoclast and that it might have been in the process of making a new lacuna.

Topics: Acid Phosphatase; Animals; Cell Nucleus; Cells, Cultured; Drug Resistance; Histocytochemistry; Image Processing, Computer-Assisted; Mice; Microscopy, Electron; Osteoclasts; Tartrates

Pellets of mineralized and demineralized bone and a composite mixture of mineralized and demineralized, devitalized bone particles were implanted subcutaneously on the dorsal body wall of young adult rats. Two weeks post-implantation, the pellets were removed and processed for histochemical and morphological analyses. Rat proximal tibia was also processed for evaluation. The levels of tartrate-resistant acid phosphatase (TRAP) activity in the multinucleated giant cells (MNGCs) from each of the three implants and from osteoclasts were assessed using an image analyzer. The osteoclasts from the proximal tibia and the majority of MNGCs from the demineralized implants demonstrated high levels of TRAP activity. MNGCs from the mineralized implants showed either a low level or absence of TRAP activity. Most MNGCs from the composite implants exhibited a low level of TRAP activity; however, there was a population of cells that demonstrated a high level of reaction product, similar to that seen in the tibia and demineralized implant. Morphologically, osteoclasts from the proximal tibia and from the osteogenic demineralized implant exhibited ruffled borders. A small population of MNGCs from the composite implant also revealed osteoclastic features. In summary, MNGCs from the mineralized implant did not exhibit a level of TRAP reaction product or morphology similar to osteoclasts, while the majority of cells from the demineralized implant and a subpopulation of the MNGCs elicited by the composite implant did demonstrate TRAP expression and morphology similar to osteoclasts. The expression of osteoclastic characteristics in cells at an ectopic site may be dependent on accessory signals from the skeletal microenvironment; such signals appear to be absent from or incomplete in the mineralized implants but appear to be present when demineralized bone particles are implanted.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Transplantation; Calcification, Physiologic; Cell Nucleus; Drug Resistance; Osteoclasts; Rats; Rats, Inbred Strains; Tartrates; Tibia; Transplantation, Heterotopic

Topics: Acid Phosphatase; Adrenal Insufficiency; Bone Remodeling; Calcium; Creatinine; Female; Humans; Hydrocortisone; Hydroxyproline; Middle Aged; Tartrates

Tartrate resistant acid phosphatase (TRAP) has been accepted as a marker for identification of osteoclasts. A method is reported here for quantitating TRAP using an image analysis system. The amount of the enzyme specific to osteoclasts can be used to differentiate osteoclasts from other cells capable of TRAP expression. TRAP expression characteristic of the osteoclast was compared with that of multi-nucleated giant cells (MNGC)s recruited to the site of subcutaneously implanted mineralized bone matrix. Two weeks post-implantation, the pellets were removed and processed for the demonstration of TRAP along with rat proximal tibiae. A large amount of TRAP was consistently expressed by the in situ osteoclasts. The MNGCs associated with the mineralized bone implants expressed little if any TRAP reaction product. Using this system, the amount of TRAP reaction product or any other enzyme reaction product expressed can be objectively and reproducibly quantitated.

Topics: Acid Phosphatase; Animals; Cytoplasm; Drug Resistance; Giant Cells; Image Processing, Computer-Assisted; Male; Osteoclasts; Rats; Reproducibility of Results; Tartrates; Tibia; Vacuoles

Serum tartrate-resistant acid phosphatase (TRAP) and total body bone mineral content (TBBM) were determined in a group of 16 children with osteogenesis imperfecta (OI) aged 5-14 years, 9 of whom suffered from type I and 7 from type III OI. TRAP and TBBM were also determined in a group of 26 normal children of a similar age range. TRAP levels were reduced in the type I and III OI groups (p less than 0.001). TBBM levels were lower in type I OI than in type III (p less than 0.005), and both OI groups showed reduced levels compared to the controls (p less than 0.001). The control group subjects showed a significant correlation between TRAP and TBBM (r = -0.62; p less than 0.001) which was not observed in the OI groups. Since TRAP is a biological marker of bone turnover, the results suggest that bone turnover is reduced in OI.

Topics: Absorptiometry, Photon; Acid Phosphatase; Adolescent; Bone Density; Child; Child, Preschool; Female; Humans; Male; Osteogenesis Imperfecta; Tartrates

In an in vivo model of osteoclastic bone resorption, we previously showed that osteocalcin-deficient bone particles (BPs), derived from warfarin-treated rats, were resorbed 50% as well as normal BPs and that they recruited fewer osteoclastic cells with decreased tartrate-resistant acid phosphatase (TRAP) activity. In order to determine the specificity of the resorption response, we evaluated the fate of implanted mixtures of normal and osteocalcin-deficient BPs. Normal and warfarin-treated donor rats were prelabeled in vivo with oxytetracycline to permit identification of BPs from either source. Normal, osteocalcin-deficient, and 50:50 mixtures of BPs (either labeled or unlabeled) were implanted into normal rats and recovered 12 days later for enzymatic (TRAP) and nondecalcified histomorphometric analyses. The incorporated oxytetracycline had no significant effect on resorption of bone particles. The recovered osteocalcin-deficient BPs were surrounded by fewer osteoclastic cells, were resorbed less, and contained less extractable TRAP activity than normal BPs. In mixed BP implants with normal and osteocalcin-deficient BPs, each type of bone particle elicited the same tissue response as when implanted separately. Remarkably, the different particles evoked dissimilar osteoclastic responses and were resorbed to different extents, even when adjacent within the same implant. These data suggest that osteocalcin may act as a substrate signal for resorption and that osteocalcin in the normal BPs does not influence the cellular response to adjacent osteocalcin-deficient BPs.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Resorption; Male; Microscopy, Electron; Osteocalcin; Osteoclasts; Oxytetracycline; Rats; Tartrates; Warfarin

Using routinely processed, paraffin-embedded tissue specimens, osteoclast-like giant cells in giant cell tumour of bone (GCT), chondroblastoma, osteoblastoma and osteoblastic osteosarcoma were examined histochemically for osteoclast-specific enzymes tartrate-resistant acid phosphatase (TRAP) and carbonic anhydrase isoenzyme II (CA-II). Osteoclast-like giant cells and some mononuclear cells possessed TRAP activity. These were further classified with respect to CA-II immunoreactivity, i.e. cells with CA-II were seen in GCT and chondroblastoma, while those in osteoblastoma and osteoblastic osteosarcoma were negative for CA-II. All the cellular components in malignant fibrous histiocytoma and various extraosseous inflammatory lesions including malignant giant cells and macrophage polykaryons were negative for both TRAP and CA-II. These results indicate that osteoclast-like giant cells in GCT, chondroblastoma, osteoblastoma and osteoblastic osteosarcoma are all osteoclasts and generated by fusion of mononuclear cells with the same histochemical characteristics as osteoclast-like giant cells. The difference in CA-II immunoreactivity suggests the functional or maturational difference between osteoclast-like giant cells in GCT and chondroblastoma and those in osteoblastoma and osteosarcoma.

Topics: Acid Phosphatase; Bone Neoplasms; Carbonic Anhydrases; Drug Resistance; Giant Cells; Histocytochemistry; Humans; Osteoclasts; Tartrates

The presence and biological activity of an Osteoclast Growth Factor (OGF) was investigated in serum-free medium conditioned by periostless fetal rat calvaria in culture. OGF activity was assessed using in vitro systems of fetal rat long bones and adult rat bone marrow cells. Rat calvaria conditioned medium (RCCM) increased the number of osteoclasts in the long bone cultures, partly due to stimulation of progenitor proliferation. RCCM did not exert a direct bone-resorbing activity (45Calcium release assay) on the pre-existing osteoclasts residing in the long bones, but stimulated bone resorption in long term cultures, apparently in an indirect manner by enhancing the number of osteoclasts. In cultures of bone marrow cells isolated from adult rats, RCCM markedly stimulated the formation of mononuclear cells which were positively stained for tartrate-resistant acid phosphatase (TRAP). The osteoclastic nature of the cells was confirmed by specific labeling with 125I-calcitonin. Formation of the TRAP-positive cells was significantly inhibited by salmon calcitonin. CM from fetal rat skin cultures did not display a significant OGF activity. Furthermore, unlike the bone marrow cells, peritoneal macrophages did not respond to RCCM and remained devoid of TRAP activity. Neutralization experiments with a specific antibody to GM-CSF indicated that OGF activity in the RCCM could not be ascribed to this hemopoietic growth factor. Secretion of OGF activity was mainly dependent on protein synthesis as addition of cycloheximide to the calvaria cultures significantly inhibited the secretion of OGF into the medium. G3000 HPLC fractionation of RCCM revealed two major OGF peaks with Mr 14,000 and 70,000. Two subsequent reverse-phase HPLC steps using the lower Mr OGF fraction led to a highly purified OGF fraction. The results of this study further provide evidence that bone tissue produces factor(s) which specifically govern the process of osteoclast development, thus providing information about one of the mechanisms controlling bone resorption.

Topics: Acid Phosphatase; Animals; Antibodies; Bone and Bones; Bone Marrow Cells; Calcitonin; Cell Division; Cells, Cultured; Culture Media; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Osteoclasts; Rats; Tartrates

A tartrate-resistant acid phosphatase activity was detected in the human placenta. This enzyme displayed immunological properties similar to those of the group of purple acid phosphatases that can be demonstrated with a rabbit polyclonal antibody against bovine spleen purple acid phosphatase. The placental enzyme was mainly localized immunohistochemically to neutrophil granulocytes of the maternal blood between the placental villi and within foetal capillaries using the bovine spleen antibody and the commercial monoclonal antibody M1 directed against an antigen found on mature granulocytes. A minor activity was detected in decidual cells and the syncytiotrophoblast. The presence of purple acid phosphatase in placental granulocytes may be related to special immunological conditions of pregnancy.

Topics: Acid Phosphatase; Granulocytes; Humans; Immunohistochemistry; Lymphocytes; Placenta; Tartrates

Human type V (tartrate-resistant) acid phosphatase belongs to a unique group of iron-binding proteins that includes uteroferrin and other purple phosphatases. The enzyme is normally restricted to osteoclasts and certain phagocytic cells but its rôle is unknown. We show that phosphatase mRNA is abundant in cells of monohistiocytic phenotype and that enzyme expression in cultured human monocyte-derived macrophages is depressed by gamma-interferon and bacterial lipopolysaccharide, agents that promote functional differentiation in these cells. In contrast, phorbol ester, which stimulates intracellular calcium-mediated events, greatly enhances type V phosphatase expression and mRNA abundance. Lymphokine and phorbol ester-modulated expression of type V acid phosphatase expression thus represents a model system for investigating proliferative responses that are specific to cells of the mononuclear macrophage system.

Topics: Acid Phosphatase; Cell Differentiation; Cells, Cultured; Gene Expression Regulation, Enzymologic; Humans; Interferon-gamma; Lipopolysaccharides; Macrophages; Monocytes; Phagocytes; RNA, Messenger; Tartrates

Inhibition of a tartrate-resistant acid phosphatase (ACP) from Leishmania donovani and the tartrate-sensitive ACP from human seminal fluid (prostatic ACP) was examined using a series of 13 molybdate-containing heteropolyanions. The heteropolyanions were divided into four groups based on the number of molybdenum atoms they contain: Group I, Mo4; Group II, Mo6-8; Group III, Mo12; Group IV, Mo18. Two of the four groups, those consisting of compounds that contain either an Mo4 unit or an Mo18 unit with a heteroatom in the central cavity, were potent inhibitors and exhibited the highest degree of selectivity against the leishmanial and seminal fluid ACPs. The inhibition of prostatic ACP by complex E2 could be completely reversed by dialysis. Little inhibition of the acid phosphatase, beta-glucuronidase, or alpha-mannosidase from human spleen was observed with complexes B' and E2. For the seminal fluid phosphatase, the Ki values obtained with arsenate and vanadate depended markedly on pH, suggesting that, unlike most other phosphatases, the conformation of the inhibitor binding site on human seminal fluid ACP is pH-dependent. Results of competition experiments performed with various inhibitor pairs indicated that complex D2 binds to the active site of prostatic ACP while complex M binds at some site on the enzyme that affects the active site. Binding of complex M also modifies the affinity of the enzyme for other inhibitors such as vanadate. The potency of several heteropolyanion complexes and their selective inhibition of pathophysiologically significant acid phosphatases indicate that these compounds may have value as tools for study of the structure and function of this class of enzyme and perhaps in the therapy of human disease.

Topics: Acid Phosphatase; Animals; Anions; Humans; Hydrogen-Ion Concentration; Kinetics; Leishmania donovani; Male; Molybdenum; Semen; Tartrates

We have reported that osteoblastic cells are required for differentiation of osteoclast progenitors in splenic tissues into multinucleated osteoclasts. In the present study we examined the pathogenesis of the osteoclast deficiency in osteopetrotic (op/op) mice using a coculture system of spleen cells and osteoblastic cells. When spleen cells obtained from op/op or normal (+/?) littermates of op/+ parent mice were cocultured with osteoblastic cells obtained from calvaria of normal ddy strain mice, numerous tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) were formed in the presence of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. Most of the TRAP-positive MNCs bound [125I]salmon calcitonin. This suggests that there is no abnormality in the osteoclast progenitors present in the splenic tissues of op/op mice. When osteoblastic cells from +/? littermates were cocultured with normal spleen cells from ddy mice, TRAP-positive MNCs were similarly formed in response to 1 alpha,25(OH)2D3. In contrast, in cocultures of op/op osteoblastic cells with normal spleen cells, no TRAP-positive cells appeared, even in the presence of 1 alpha,25(OH)2D3. The op/op mutation was recently reported to exist in the coding region of the macrophage colony-stimulating factor (M-CSF) gene. Adding M-CSF and 1 alpha,25(OH)2D3 to the coculture with op/op osteoblastic cells induced the appearance of TRAP-positive MNCs with calcitonin receptors. These results clearly indicate that osteoclast deficiency in op/op mice is due to a defect in the local microenvironment in bone, in which M-CSF produced by osteoblastic cells plays a critical role in osteoclast development.

Topics: Acid Phosphatase; Animals; Calcitonin; Calcitriol; Cell Differentiation; Cells, Cultured; Female; Macrophage Colony-Stimulating Factor; Male; Mice; Osteoblasts; Osteoclasts; Osteopetrosis; Spleen; Stem Cells; Tartrates

Gallium nitrate is a clinically effective agent for the treatment of cancer related hypercalcemia. The mechanism of action of this agent was investigated following development of a quantitative in vivo bone resorption assay modified from the method of Glowacki. In a preliminary study, the time course of resorption of 50 mg subcutaneous implants of bone powder in growing rats was followed by chemical analysis of mineral (ash and Ca) contents, enzymatic and histochemical assay of tartrate resistant acid phosphatase (TRAP) activity, and image analysis of changes in particle size using von Kossa stained sections. Day 21 was chosen as a single time point for the comparison of the extent of resorption of gallium-containing and control bone particles. Resorption of bone particles containing 0.39 micrograms Ga/mg bone was significantly inhibited relative to control particles. Mineral content (6.7 vs. 3.6 mg), Ca content (1.72 vs. 1.37 mg), and the percentage of the field covered by bone particles (12 vs. 9%) were greater in the animals which received gallium-containing bone particles. Similarly, the number of osteoclast-like cells and the TRAP activity in the gallium-containing bone particle implants at 21 days were increased relative to controls. These data indicate that gallium incorporation into bone matrix confers resistance to resorption.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Density; Bone Matrix; Bone Resorption; Calcium; Gallium; Hypercalcemia; Male; Osteoclasts; Particle Size; Rats; Rats, Inbred Strains; Tartrates

A potent inhibitor of carbonic anhydrase, 5-[3-hydroxybenzoyl]thiophene-2-sulfonamide (HTS), was shown to cause a 37% reduction in the area of resorption pits formed by isolated chick osteoclasts when used at a dose of 10(-7) M. HTS at doses of 10(-9) and 10(-7) M was also effective in reducing acid formation by the osteoclasts (14 and 36%, respectively). Additionally, the effect of HTS was found to be readily reversed by removing the agent, showing that it does not exert a toxic effect on the cells. This study indicates that the inhibitory effect of HTS on bone resorption is at the level of the acid-forming mechanism in osteoclasts and supports the view that carbonic anhydrase has a central role in the process.

Topics: Acid Phosphatase; Acridine Orange; Animals; Bone Resorption; Carbonic Anhydrase Inhibitors; Cells, Cultured; Chickens; Osteoclasts; Tartrates; Thiophenes

The method of Barka and Anderson was used for the demonstration of tartrate-resistant acid phosphatase (TRAcP) in cultures of bone marrow, spleen, lung, and peritoneal cells of the mouse. The staining was performed either in the usual way by adding both substrate (naphthol-AS-BI-phosphate) and coupler (hexazonium pararosanilin) together (the simultaneous-coupling technique) or by adding first the substrate and then the coupler (the post-coupling technique). We measured TRAcP-activity fluorometrically after extraction of the product naphthol-AS-BI, using the same staining solution as in cytochemical method, but without the coupler. In bone marrow, spleen, lung, and peritoneal cell cultures a biochemically measurable TRAcP-activity was detected. Post-coupling generally gave a higher level of staining and larger numbers of TRAcP-positive cells than simultaneous-coupling. In bone marrow cultures macrophages, identifiable by their ability to phagocytose microspheres, became TRAcP-positive during culture. In lung cell cultures cells capable of phagocytosis of bacteria were shown to be TRAcP-positive. Peritoneal macrophages remained TRAcP-negative in the simultaneous-coupling technique. Using the post-coupling technique a small number stained TRAcP-positive. In spleen cell cultures TRAcP-positive cells containing hemosiderin were visible. In cultures of all four cell types, F4/80 positive cells staining also for TRAcP were present. F4/80 is a well known marker for macrophages, whereas osteoclasts are negative. In conclusion, mouse macrophages originating from various tissues can become TRAcP-positive in vitro. TRAcP activity alone is not a reliable marker for osteoclasts in bone marrow cultures.

Topics: Acid Phosphatase; Animals; Biomarkers; Bone Marrow; Bone Marrow Cells; Cells, Cultured; Fluorometry; Lung; Macrophages; Male; Mice; Osteoclasts; Peritoneal Cavity; Phagocytosis; Spleen; Tartrates

Bioptic material from the spleen of a three-year-old boy with a type 1 Gaucher disease was studied by immuno- and cytochemical methods with special regard to the macrophage-derived Gaucher cells. These cells were positive with PAS and Prussian blue staining, and were immuno-positive with the monoclonal 25 F9 antibody, specific to mature, non-inflammatory macrophages. Large Gaucher cells and their postulated small precursor cells revealed strong tartrate-resistant acid phosphatase (TRAP) and unspecific carboxylate esterase activities. Using a polyclonal antibody to bovine spleen purple phosphatase, a lysosomal TRAP from splenic macrophages, the TRAP of the Gaucher cells could be identified to belong to this group of iron-containing, purple acid phosphatases immunocytochemically. The origin of splenic Gaucher cells from blood monocytes and their further development are discussed.

Topics: Acid Phosphatase; Child, Preschool; Gaucher Disease; Humans; Immunohistochemistry; Male; Spleen; Tartrates

Demineralized bone matrix contains a number of growth factors for osteoblast-like cells. Two of these, the novel glycoprotein osteoinductive factor (OIF) and transforming growth factor-beta (TGF beta), act together to cause ectopic bone formation in vivo. Since OIF, like TGF beta, is likely released from bone when the matrix is resorbed, we examined the effects of homogeneous OIF and TGF beta on osteoclast function. Osteoclast function was tested in isolated avian osteoclasts and was measured in terms of tartrate-resistant acid phosphatase (TRAP) activity, oxygen-derived free radical production, and formation of characteristic resorption lacunae on slices of sperm whale dentine. OIF (50-100 ng/ml) inhibited the capacity of these osteoclasts to form lacunae whether assessed by the number of excavations per slice or by the total area resorbed. OIF (10-100 ng/ml) or TGF beta (10-20 ng/ml) caused a decrease in TRAP activity as well as a reduction in oxygen-derived free radical generation detected by nitroblue tetrazolium staining. TGF beta had no effect on the resorption capacity of isolated osteoclasts in concentrations that inhibited TRAP activity and nitroblue tetrazolium staining. These results suggest that growth regulatory factors, such as OIF and TGF beta, released during the resorption of bone may be endogenous inhibitors of continued osteoclastic activity. This cessation of osteoclast activity may be an essential preliminary step to the new bone formation that occurs at resorption sites during bone remodeling.

Topics: Acid Phosphatase; Animals; Bone Matrix; Bone Resorption; Cattle; Chickens; Free Radicals; Glycoproteins; Growth Substances; Nitroblue Tetrazolium; Osteoclasts; Oxygen; Staining and Labeling; Tartrates; Transforming Growth Factors

In order to evaluate the usefulness of a recently described acid ATPase as a marker for osteoclast differentiation, we have performed histochemical and biochemical analyses of the distribution of tartrate-resistant acid phosphatase (TRAP) and tartrate-resistant acid ATPase (TrATPase) in bone, bone marrow and spleen. Histochemical studies of bone demonstrated that multinucleated osteoclasts stained for both TRAP and TrATPase. However, staining for TRAP covered the entire cytoplasm, whereas TrATPase staining was localized primarily to cytoplasmic areas next to bone and on adjacent mineralized surfaces. Occasionally TrATPase-positive mononuclear cells were observed on excavations in the bone surface. In the spleen, mononuclear TRAP-positive cells were located in the marginal zone between the white and red pulp, whereas no staining for TrATPase was observed. Comparison of the biochemically measured TRAP and TrATPase activities showed that bone had the highest specific activity for both enzymes followed by the bone marrow and spleen. However, enzyme activity in the spleen compared to bone was about 4-fold higher for TRAP compared to TrATPase. Additional evidence for a restricted expression of TrATPase to bone relative to spleen was obtained by in vitro translation studies. These data indicate that TrATPase is a more selective marker than TRAP in histochemical and biochemical studies of osteoclast differentiation and furthermore suggest that development of TrATPase is a late acquisition in osteoclast ontogeny.

Topics: Acid Phosphatase; Adenosine Triphosphatases; Animals; Bone and Bones; Bone Development; Bone Marrow; Cell Differentiation; Cytoplasm; Histocytochemistry; Osteoclasts; Rats; Spleen; Tartrates

Osteoclast-like multinucleated giant cells are induced in bone marrow cultures by 1,25-dihydroxyvitamin D3 and other agents. These cells resemble osteoclasts in their morphology, their ability to resorb bone, and the possession of calcitonin receptors. We report here a biphasic effect of transforming growth factor-beta (TGF beta) on the generation of these cells in mouse bone marrow cultures. At low concentrations (10-100 pg/ml) TGF beta enhanced 1,25-dihydroxyvitamin D3-dependent production of osteoclast-like cells, while at higher concentrations TGF beta was inhibitory. Complete inhibition was seen at 4 ng/ml. Antibodies directed against TGF beta significantly reduced the generation of osteoclast-like cells in 1,25-dihydroxyvitamin D3-treated cultures, indicating the contribution of endogenous TGF beta activity. TGF beta also enhanced the accumulation of prostaglandin E2 (PGE2) in a dose-dependent manner. Moreover, we found that the generation of these cells in response to 1,25-dihydroxyvitamin D3 was also dependent on PG accumulation, since it was inhibited by indomethacin (250 ng/ml), and this inhibition could be reversed by exogenous PGE2. It is, thus, suggested that PG activity, probably PGE2, mediates the enhancing effect of low TGF beta concentrations and is required for the generation of osteoclast-like cells in this system.

Topics: Acid Phosphatase; Animals; Antibodies; Bone Marrow; Bone Marrow Cells; Bone Resorption; Calcitriol; Cells, Cultured; Dinoprostone; Indomethacin; Male; Mice; Mice, Inbred C57BL; Osteoclasts; Receptors, Calcitonin; Receptors, Cell Surface; Tartrates; Transforming Growth Factors

Osteoclast development was studied in cell cultures prepared from calvaria of neonatal osteopetrotic (mi/mi) mice or their normal littermates, using tartrate-resistant acid phosphatase (TRAPase), as an osteoclast marker. In cultures from normal mice, treatment with 10 nM PTH for 4-5 days stimulated the formation of osteoclasts. However in cultures from mi/mi mice, this response was only 7% +/- 5% that of normal mice and they were significantly smaller than osteoclasts of normal mice. Mineralized bone particles elicited osteoclast development in cultures from both normal and mi/mi mice, and osteoclast size was identical for both genotypes. Seventy-eight to 96% of the TRAPase-positive cells bound 125I-CT, as demonstrated by autoradiography. 125I-CT binding characteristics were identical in cultures from both genotypes treated with bone particles, exhibiting a Kd of 3.3-3.6 x 10(-10) M. Addition of PTH stimulated 45Ca release from the added bone particles only in the case of cultures prepared from normal mice, and CT inhibited this response. Cells from normal mice were capable of excavating bone from the surface of smooth cortical bone wafers, but such excavations were rarely seen in the case of calvarial cells from mi/mi mice. Thus, PTH-driven differentiation of osteoclasts is arrested in calvarial cell cultures from mi/mi mice, but mi/mi preosteoclasts retain the ability to express certain osteoclast markers in response to bone derived signals. We hypothesize that the lack of activity of mi/mi osteoclasts is due to the failure of mi/mi preosteoclasts to respond appropriately to resorptive agents, or to cytokines elicited by these agents.

Topics: Acid Phosphatase; Animals; Bone and Bones; Calcitonin; Cell Differentiation; Cells, Cultured; Kinetics; Mice; Osteoclasts; Osteopetrosis; Parathyroid Hormone; Protein Binding; Tartrates

In search for invariant surface proteins in Trypanosoma brucei bloodstream forms, acid phosphatase was investigated. Earlier work had shown that part of the cellular phosphatase activity is associated with the flagellar pocket of the parasite. It is demonstrated that T. brucei contains at least two membrane-bound enzymes, one is sensitive to the inhibitor L-(+)-tartrate while the other is resistant. The tartrate-sensitive phosphatase was purified to homogeneity by monoclonal antibody affinity chromatography and shown to be a glycoprotein of low abundance (13,000 molecules/cell). It has an apparent molecular weight of 70,000 Da. The usefulness of acid phosphatase as a marker for characterizing the membrane lining the flagellar pocket is discussed.

Topics: Acid Phosphatase; Animals; Membrane Proteins; Mice; Mice, Inbred BALB C; Microscopy, Immunoelectron; Sensitivity and Specificity; Tartrates; Trypanosoma brucei brucei

The influence of mechanical stimulation by intermittent compressive force (ICF) of physiologic magnitude on osteoclastic bone resorption was investigated in cultures of fetal mouse cartilaginous long bones. Exposure to ICF resulted in a significant decrease in mineral resorption, as indicated by the decreased release of 45Ca and a decreased number of osteoclasts in the diaphysis. Conditioned medium (CM) from ICF-exposed periosteum-free cultures (ICF-CM), but not from control cultures (Co-CM), inhibited mineral resorption in fresh bones cultured under control conditions. Co-CM increased, but ICF-CM decreased, the number of tartrate-resistant acid phosphatase-positive cells in 7-day bone marrow cultures. Direct exposure of bone marrow cultures to ICF yielded the same results. Thus, osteoclastic bone resorption in cartilaginous long bones is inhibited by ICF in vitro. A soluble factor(s) acting on tartrate-resistant acid phosphatase-positive, osteoclast precursor-like cells seems to play a role in this effect.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Resorption; Culture Media; Drug Resistance; Mice; Minerals; Organ Culture Techniques; Osteoclasts; Physical Stimulation; Pressure; Tartrates

In order to explore why ovarian hormone deficiency causes excessive osteoclastic bone resorption that results in osteoporosis in a large number of postmenopausal women, bone marrow cells from ovariectomized and sham-operated female mice were cultured for 8 days. The cells gave rise in culture to tartrate-resistant acid phosphatase-positive multinucleate cells. The formation of these osteoclast-like cells was enhanced by parathyroid hormone and 1,25(OH)2vitamin D3, with the latter being more effective. Cultures of cells from ovariectomized animals formed significantly more tartrate-resistant acid phosphatase-positive multinucleate cells than those from sham-operated controls. These findings support the hypothesis that ovarian hormone deficiency promotes the expansion of a pool of marrow-derived progenitor cells that differentiate into bone-resorbing osteoclasts under the influence of osteotropic hormones.

Topics: Acid Phosphatase; Animals; Bone Marrow Cells; Calcitriol; Cell Division; Cells, Cultured; Female; Mice; Mice, Inbred ICR; Osteoclasts; Ovariectomy; Ovary; Parathyroid Hormone; Tartrates

The influence of androgen on prostate differentiated cell function was investigated using primary cultures of rat ventral prostate epithelial and stromal cells developed from sexually immature animals (21 days of age). As a biochemical marker of androgen action, total acid phosphatase activity, which comprises both the secretory and lysosomal isoforms, was measured. Testosterone increased total acid phosphatase activity approximately 2-fold in epithelial cell cultures. This increase occurred only after the cessation of cell proliferation (i.e. upon reaching a confluent monolayer). In contrast, stromal cells showed no significant change in total acid phosphatase activity in response to androgen. Polyacrylamide gel isoelectric focusing of total acid phosphatase activity from epithelial and stromal cell extracts revealed that secretory acid phosphatase activity was localized exclusively in the epithelial cells while lysosomal acid phosphatase activity was present in both cell types. Furthermore, the androgen-induced increases in epithelial total acid phosphatase activity were found to result from increases in the secretory isoform.

Topics: Acid Phosphatase; Animals; Cells, Cultured; Epithelium; Isoelectric Focusing; Isoenzymes; Male; Prostate; Rats; Rats, Inbred Strains; Tartrates; Testosterone; Tissue Distribution

Multinucleated cells containing tartrate-resistant acid phosphatase were produced in mouse bone marrow cultures in response to 1,25-dihydroxyvitamin D3. These cells resemble osteoclasts in their morphology, possess receptors for calcitonin, and resorb bone in culture. The effects of several hemopoietic regulatory proteins on the generation of these cells were examined in this study. Interleukin-3, granulocyte-macrophage-stimulating factor (GMCSF), and macrophage-stimulating factor strongly inhibited generation of the tartrate-resistant acid phosphatase-containing multinucleated cells with approximate EC50 values of 3, 6, and 3 colony-forming units/ml, respectively. Granulocyte colony stimulating factor, interleukin-6, and leukemia inhibitory factor had no effect on the generation of these cells. In addition, we observed that the number of these cells was reduced when the bone marrow was plated at high cell density, and that this inhibitory effect was reversed by the addition of neutralizing antibodies directed against GMCSF. These findings suggest that GMCSF and other hemopoietic factors secreted by cells in the bone marrow regulate development of the osteoclast-like cells, possibly by diverting common precursor cells to alternate pathways.

Topics: Acid Phosphatase; Animals; Antibodies; Bone Marrow; Bone Marrow Cells; Calcitonin; Cell Count; Cell Division; Cells, Cultured; Colony-Stimulating Factors; Drug Resistance; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Hematopoiesis; Mice; Osteoclasts; Tartrates

We present a modified histochemical method for staining osteoclasts and adjacent mononuclear cells which takes advantage of the recently described substrate specificity for ATP of osteoclastic acid phosphatase. Staining of osteoclasts using ATP as substrate exhibits by light microscopy the same tartrate resistance as conventional acidic phosphatases, without the bone surface staining seen with other substrates. This feature, coupled with specific staining of fewer vicinal mononuclear cells, makes this method potentially useful for studying osteoclast ontogeny and function.

Topics: Acid Phosphatase; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Histocytochemistry; Osteoclasts; Rats; Staining and Labeling; Tartrates

beta-TCP was implanted in surgically prepared alveolar bone defects on the mesial side of the upper canine. The dogs that we used were sacrificed after 5 weeks, fixed by perfusion, and the beta-TCP resorbing cells were examined ultrastructurally and histochemically, with the following results: (1) beta-TCP was resorbed by macrophages and multinucleated giant cells. (2) Mitochondria, vacuoles and Golgi apparatus were abundant in beta-TCP-resorbing multinucleated giant cells that possessed neither ruffled borders nor clear zones. (3) The addition of tartric acid inhibited acid phosphatase activity in the cytoplasm of the multinucleated giant cells and macrophages.

Topics: Acid Phosphatase; Animals; Calcium Phosphates; Dental Implants; Dogs; Giant Cells; Histocytochemistry; Macrophages; Microscopy, Electron; Osteoblasts; Periodontium; Tartrates

Acid phosphatases were examined histochemically at the light- and electron-microscopic levels using para-nitrophenyl phosphate (pNPP) and beta-glycerophosphate (beta-GP) as substrates. By light microscopy, there was intense activity with pNPP in supranuclear and distal regions of the secretory ameloblast, and moderate or slight activity respectively in those regions with beta-GP. These enzyme activities were less at the late secretory stage of amelogenesis and disappeared at the transitional stage. By electron microscopy, acid phosphatase activity was seen in the trans side cisternae of the Golgi apparatus, in lysosome-like granules, and in small vesicles in the Tomes' processes. The activity with pNPP but not beta-GP was also localized at the plasma membrane (proximal, lateral and distal surface). Activity with beta-GP was completely inhibited by 1 mM sodium tartrate and by 1 mM NaF; activity with pNPP was inhibited by 1 mM NaF and 10 mM sodium tartrate, but not by 1 mM sodium tartrate. Thus there are at least two different acid phosphatases, one tartrate-sensitive and the other 1 mM tartrate-resistant, in the secretory ameloblast; the tartrate-resistant enzyme is plasma-membrane bound.

Topics: Acid Phosphatase; Ameloblasts; Amelogenesis; Animals; Cell Membrane; Cytoplasmic Granules; Glycerophosphates; Golgi Apparatus; Histocytochemistry; Microscopy, Electron, Scanning; Molar; Nitrophenols; Organophosphorus Compounds; Rats; Rats, Inbred Strains; Tartrates; Tooth Germ; Vacuoles

Tartrate-resistant acid phosphatase activity (TRAPase) is widely used as a cytochemical marker to distinguish osteoclasts from macrophages and other related cell types. The degree of tartrate resistance, however, may depend on which reaction methods, tissues, or species are used. To investigate this further, we have measured the amount of cytochemical reaction product by microdensitometry. We compared osteoclast acid phosphatase (APase) activity in fresh frozen sections of neonatal rat calvaria using two different reaction methods; one is commonly employed for qualitative histochemistry and includes alpha naphthyl phosphate as substrate, simultaneous coupling to the chromagen Fast Garnet, and a 30-minute reaction time (method A). The other may be used to measure enzyme reaction rates in cells in situ and employs conditions suitable for initial velocity kinetics, namely naphthol-ASBI phosphate as substrate, post coupling to Fast Garnet, and a 2-minute reaction time. Although enzyme reaction rates differed greatly between the two methods, significant inhibition of APase activity by tartrate was observed in calvarial osteoclasts (69% and 59% with methods A and B, respectively), osteoblasts, and spleen macrophages. Using method B, mouse calvarial osteoclasts had similar APase activity to that seen in the rat. Tartrate produced little inhibition in these mouse cells, in contrast to the observations made with rat tissue, but murine spleen macrophages were significantly tartrate sensitive (40% inhibition with tartrate). On this basis, conclusions regarding the cell specificity of TRAPase should be treated cautiously.

Topics: Acid Phosphatase; Animals; Bone and Bones; Densitometry; Histocytochemistry; Macrophages; Mice; Osteoclasts; Rats; Spleen; Tartrates

Tartrate-resistant acid phosphatases types 5a and 5b were purified from human hairy cell leukemia spleen by sequential chromatography on Phenyl-Sepharose, CM-Sepharose, concanavalin A-Sepharose, FPLC Superose-12 and FPLC Mono-S. The purification over the original tissue extract was 1150- and 3300-fold, with a yield of 2.1% and 2.5%, respectively. Gel filtration indicated an Mr of about 30000 for both forms. There was a N-terminal sequence identity between the two enzymes. However, they appeared to be different as assessed by cation exchange chromatography and amino acid composition.

Topics: Acid Phosphatase; Amino Acid Sequence; Amino Acids; Chromatography; Chromatography, High Pressure Liquid; Drug Resistance; Humans; Leukemia, Hairy Cell; Male; Middle Aged; Molecular Sequence Data; Molecular Weight; Spleen; Tartrates

Tartrate resistant acid phosphatase (TRAP) is considered as an enzyme marker for osteoclasts and their precursors. A cytomorphometric study of the osteoclastic population was made after TRAP staining on transiliac bone biopsies from 10 human volunteers, and the diameter of each TRAP positive profile was determined with an automatic image analyser. The frequency distribution of the cell diameter followed a lognormal law. TRAP positive cells along bone trabeculae belong to a homogeneous osteoclastic population.

Topics: Acid Phosphatase; Adult; Histocytochemistry; Humans; Male; Osteoclasts; Tartrates

After our previous report that osteoclast-like multinucleated cells (MNCs) were formed in response to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] in cocultures of mouse spleen cells and osteoblast-rich populations freshly isolated from fetal mouse calvariae, we examined whether such primary osteoblast-like cells can be replaced by established cell lines in inducing osteoclast-like cell formation. We first used two clonal cell lines simultaneously established from newborn mouse calvariae. One was the osteoblastic cell line MC3T3-E1, and the other was the preadipose cell line MC3T3-G2/PA6. Tartrate-resistant acid phosphatase (TRACP; a marker enzyme of osteoclasts)-positive mononuclear cells and MNCs were formed in the cocultures of spleen cells and MC3T3-G2/PA6 cells in the presence of 1 alpha,25-(OH)2D3. Dexamethasone greatly potentiated TRACP-positive MNC formation induced by 1 alpha,25-(OH)2D3, whereas the glucocorticoid alone had no effect on it. In contrast, osteoblastic MC3T3-E1 cells failed to induce TRACP-positive cells in the cocultures. Another bone marrow-derived stromal cell line ST2 also induced TRACP-positive MNC formation in the cocultures in response to 1 alpha,25-(OH)2D3 and dexamethasone. Salmon calcitonin enhanced cAMP production in the cocultures only when TRACP-positive cells were formed. Autoradiographic studies demonstrated that [125I]calcitonin specifically bound to TRACP-positive cells formed in the cocultures. When spleen cells and either MC3T3-G2/PA6 or ST2 cells were cocultured on sperm whale dentine slices in the presence of 1 alpha,25-(OH)2D3 and dexamethasone, numerous resorption lacunae were formed. These results show that the two bone marrow-derived stromal cell lines can support osteoclast-like cell differentiation in cocultures with spleen cells.

Topics: Acid Phosphatase; Animals; Autoradiography; Bone Marrow Cells; Calcitonin; Calcitriol; Cell Differentiation; Cell Division; Cell Line; Cytological Techniques; Dexamethasone; Drug Resistance; Mice; Osteoclasts; Spleen; Tartrates

Rat and chicken osteoclasts were cultured on bone slices, where they showed active resorption with resorption lacunae, which cold be seen by toluidine blue staining or with a scanning electron microscope. Osteoclast microfilaments, F-actin, vinculin, and talin were studied by immunofluorescence. In attached osteoclasts, vinculin appeared as a double circle in the periphery of the cell, and the most intense F-actin staining was located between these vinculin zones. Some chicken osteoclasts showed also intense F-actin staining throughout the center of the cell. Talin appeared in a similar double circle to vinculin. This kind of distribution of microfilaments was always associated with resorption lacunae, and F-actin, vinculin, and talin zones correspond roughly to the edge of lacunae. Osteoclasts showing a diffuse staining pattern were not associated with a resorption pit. The results suggest that this specific microfilament arrangement is located at the attachment zone of the osteoclast and is obligatory for the attachment and resorption. However, this arrangement of microfilaments is quite different from the one that has been previously described in osteoclasts cultured on glass.

Topics: Acid Phosphatase; Actin Cytoskeleton; Animals; Antibodies, Monoclonal; Bone Resorption; Cell Adhesion; Cells, Cultured; Chickens; Culture Techniques; Cytoskeleton; Fluorescent Antibody Technique; Osteoclasts; Rats; Tartrates

Tartrate-resistant acid phosphatase (TRAP) is a histochemical marker for osteoclasts, the multinucleated bone resorbing cell. This type 5 acid phosphatase has been purified 500-fold from human bone by three chromatographic steps: cation exchange, gel filtration, and HPLC cation exchange. Like most other TRAPs isolated, it is a basic glycoprotein of a molecular weight about 33,000. Its pH optimum Km, and Vmax for p-nitrophenyl phosphate are 5.7, 0.8 mM, and 12 units/mg, respectively. Human bone TRAP hydrolyzes aryl phosphates, nucleoside di- and triphosphates, pyrophosphate, and phosphoproteins. It is activated by mild reducing agents but inhibited by molybdate, fluoride, arsenate, phosphate, and dithionite. Its activity is not inhibited by tartrate, a feature that distinguishes it from other acid phosphatases. Sodium etridonate, the bisphosphonate used clinically to reduce bone resorption, is a relatively poor inhibitor of bone TRAP. Human bone TRAP is immunologically related to the porcine uterine secretory TRAP, uteroferrin. Monospecific rabbit antibodies to the bone TRAP have been immunopurified by using affinity chromatography with uteroferrin immobilized on Sepharose and can be used to detect low amounts of the enzyme in a simple dot-blot assay.

Topics: Acid Phosphatase; Biomarkers; Bone and Bones; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Humans; Hydrogen-Ion Concentration; Hydrolysis; Immunochemistry; Molecular Weight; Substrate Specificity; Tartrates

For identification of semen in stain the specific activity of L-tartrate-inhibitable acid phosphatase (ACP) was determined. With each stain extract, both enzyme activity and protein concentration were determined, and the specific activity (enzyme activity/protein concentration) was calculated. Seminal stains showed a value of 23.8 +/- 15.2 (mean +/- SD) IU/mg protein, while vaginal fluid stains showed a value of 0.088 +/- 0.049 IU/mg protein. Stains of other body fluids did not show any L-tartrate-inhibitable ACP activity. Furthermore, only eight of 30 plant juice stains showed any levels of L-tartrate-inhibitable ACP, although all plants tested showed ACP activity. As the present method enables us to analyze forensic samples quantitatively, it seems to be useful for forensic practice.

Topics: Acid Phosphatase; Hemagglutination Inhibition Tests; Humans; Male; Rape; Semen; Tartrates

Lysosomes are defined traditionally with the marker enzyme acid phosphatase. We showed recently that lysosomes from human fibroblasts can be separated into a light and dense fraction as well as prelysosomal population. We now provide evidence that although acid phosphatase is enriched in all three fractions, the marker enzyme in the prelysosomal compartment is qualitatively distinct from that of the lysosomes. Ultrastructural analysis showed that the acid phosphatase in the prelysosomal vesicles deposited an extremely electron-dense reaction product, entirely obliterating the lumen of the vesicle, in contrast to that of the light and dense lysosomes which deposited a fine and diffuse product scattered throughout the luminal space. Biochemical analysis showed that only 51% of the acid phosphatase in the prelysosomes was inhibited by tartrate, while 80% of that in the lysosomes was tartrate-inhibitable. Immunoprecipitation with antibodies specific for various isozymes of acid phosphatase showed that 39% of the acid phosphatase in the prelysosomes was of the 'lysosomal' type whereas over 50% of the acid phosphatase in the lysosomes was of this type. These results showed that acid phosphatase in the prelysosomes of human cultured fibroblasts can be distinguished from that of the lysosomes cytochemically, biochemically, and immunologically and that lysosomes, as marked by acid phosphatase, are a heterogeneous organelle.

Topics: Acid Phosphatase; Cell Fractionation; Cells, Cultured; Fibroblasts; Humans; Intracellular Membranes; Isoenzymes; Lysosomes; Precipitin Tests; Tartrates

Topics: Acid Phosphatase; Adolescent; Adult; Aged; Alkaline Phosphatase; Bone and Bones; Child; Child, Preschool; Female; Humans; Isoenzymes; Male; Middle Aged; Reference Values; Tartrates

We describe an immunoassay for human band-5 acid phosphatase in which antibodies to porcine uteroferrin, immobilized on Sepharose particles, are used. Band-5 acid phosphatase is the tartrate-resistant isoenzyme normally expressed in tissue macrophages such as osteoclasts and alveolar macrophages. The immunoassay is similar in reproducibility and sensitivity to assays based on inhibition by d-tartrate. However, compared with the latter, the greater specificity of the immunoassay makes it markedly less susceptible to errors arising from the presence of non-band-5 acid phosphatases, e.g., from prostate.

Topics: Acid Phosphatase; Animals; Antibodies; Humans; Immunoassay; Isoenzymes; Macrophages; Male; Metalloproteins; Prostate; Pulmonary Alveoli; Quality Control; Reference Values; Spleen; Swine; Tartrate-Resistant Acid Phosphatase; Tartrates

We developed a co-culture system with mouse spleen cells and osteoblastic cells to examine the role of osteoblasts in osteoclast formation. When mouse spleen cells and osteoblastic cells isolated from fetal mouse calvariae were co-cultured in the presence of 10 nM 1 alpha, 25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], numerous tartrate-resistant acid phosphate (TRACP)-positive mononuclear and multinucleated cells were formed within 8 days. Neither the same co-cultures without the vitamin nor separate cultures of either spleen cells or osteoblastic cells with the vitamin produced TRACP-positive cells. Salmon calcitonin (CT) markedly increased cAMP production in the co-cultures treated with 1 alpha,25(OH)2D3. Autoradiographic studies clearly demonstrated that [125I]-CT specifically bound to the TRACP-positive cells formed in the co-cultures with the vitamin. When spleen cells and osteoblastic cells were co-cultured on dentine slices in the presence of 1 alpha,25(OH)2D3, numerous resorption lacunae were formed on the slices. Neither co-cultures of alveolar macrophages and osteoblastic cells nor those of spleen cells and mouse skin-derived fibroblasts induced TRACP-positive cells even in the presence of 1 alpha,25(OH)2D3. When spleen cells and osteoblastic cells were cultured separately from each other by a membrane filter (0.45 micron), no TRACP-positive cells were formed. These results indicate that osteoblastic cells are required for the differentiation of osteoclast progenitors in splenic tissues into multinucleated osteoclasts.

Topics: Acid Phosphatase; Animals; Calcitonin; Calcitriol; Cell Differentiation; Cells, Cultured; Cyclic AMP; Embryo, Mammalian; Mice; Microscopy, Electron, Scanning; Osteoblasts; Osteoclasts; Spleen; Stem Cells; Tartrates

Bone-induced multinucleated cells have been suggested as surrogates for the study of osteoclastic lineage and function. This study evaluates this proposal by comparing acid phosphatase localization in tibial osteoclasts (in situ) with that of cell populations elicited by subcutaneous implantation of devitalized trabecular bone chips from two week old rats and suture into normal and osteopetrotic (ia) rats, emphasizing tartrate-resistant acid phosphatase, an osteoclastic marker. The ia rat mutation of osteopetrosis is characterized by defective osteoclasts which typically express enhanced TRAP activity when compared to normal; ia macrophage populations do not share the same osteoclastic defect and demonstrate normal amounts of acid phosphatase reactivity. The majority of the acid phosphatase activity expressed by implant-elicited mononuclear cells was tartrate sensitive. An increase in the percentage of tartrate-sensitive, but not TRAP-positive, mononuclear cells was observed during the 14-day implantation period, suggesting the mononuclear cells did not undergo osteoclastic differentiation. Both normal and ia osteoclasts contained high concentrations of TRAP reaction product (++) while bone- and suture-induced multinucleated cells examined at 14 days post-implantation were negative (0) or mildly (+) TRAP reactive. We conclude that devitalized bone matrix implanted at this ectopic site is capable of the formation of TRAP-positive (+) multinucleated cells, but when compared on the basis of strength of TRAP activity, the bone-induced multinucleated cells do not resemble active osteoclasts, but are similar to suture-elicited macrophage polykaryons. Therefore, we suggest caution in the use of bone-induced multinucleated cells as surrogates for the study of osteoclasts and normal bone resorption. Instead, these cells may represent a population of cells involved in pathological bone loss due to inflammation.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Transplantation; Histocytochemistry; Insect Proteins; Macrophages; Osteoclasts; Osteopetrosis; Proteins; Rats; Rats, Mutant Strains; Silk; Sutures; Tartrates; Tibia

We developed a mouse bone marrow culture system to examine the process of osteoclast-like multinucleated cell formation from its progenitors. When mouse marrow cells were cultured for 8 days with 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3, 10(-10) to 10(-7) M] or human PTH (1-34) (25-100 ng/ml), tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells formed. No TRACP-positive multinucleated cells appeared in the absence of these hormones. 1 alpha,25-(OH)2D3 and PTH also increased the number of the clusters of TRACP-positive mononuclear cells. Time course studies showed that these TRACP-positive mononuclear cell clusters appeared before the formation of TRACP-positive multinucleated cells, suggesting that the TRACP-positive mononuclear cells are precursors of the multinucleated cells. Salmon calcitonin markedly inhibited the formation of TRACP-positive multinucleated cells but not TRACP-positive mononuclear cell clusters induced by 1 alpha,25-(OH)2D3 or PTH. TRACP-positive mononuclear cells and multinucleated cells were rarely stained for nonspecific esterase, but some mononuclear cells were positively stained for both nonspecific esterase and TRACP. More that 90% of the TRACP-positive mononuclear cell clusters and multinucleated cells were found near colonies of alkaline phosphatase-positive mononuclear cells (possibly osteoblasts). When marrow mononuclear cells were cultured on sperm whale dentine slices in the presence of 1 alpha,25-(OH)2D3 or PTH, numerous resorption lacunae were formed. These results suggest that 1) TRACP-positive multinucleated cells formed in response to osteotropic hormones in mouse marrow cultures satisfy most of the criteria of osteoclasts, and 2) osteoblasts may play an important role in osteoclast formation.

Topics: Acid Phosphatase; Animals; Bone Marrow Cells; Calcitonin; Calcitriol; Cells, Cultured; Male; Mice; Microscopy, Electron, Scanning; Osteoclasts; Parathyroid Hormone; Tartrates

We studied several factors affecting biological variation in serum acid phosphatases in a population of 1195 apparently healthy subjects four years old or older. We assayed total acid phosphatase activities in the presence of a transphosphorylating agent and using alpha-naphthyl phosphate as substrate. The main factors modifying total and tartrate-resistant acid phosphatases activities in serum are similar to those observed for total and bone alkaline phosphatases activities: age, sex, and hormonal state (puberty or menopause). The tartrate-inhibited acid phosphatase activity is, however, independent of biological variations. Finally, we propose reference limits for total, tartrate-resistant, and tartrate-inhibited acid phosphatases in serum.

Topics: Acid Phosphatase; Adolescent; Adult; Age Factors; Child; Child, Preschool; Female; Humans; Menarche; Menopause; Middle Aged; Reference Standards; Sex Factors; Tartrates

Fifty two of 63 patients with renal osteodystrophy had one or more mononuclear cells positive for acid phosphatase in the marrow. These cells are also tartrate resistant and non-specific esterase negative, and are believed to be precursors to osteoclasts and other acid phosphatase positive cells resorbing bone on the trabecular surface.

Topics: Acid Phosphatase; Bone Marrow; Bone Resorption; Carboxylesterase; Carboxylic Ester Hydrolases; Chronic Kidney Disease-Mineral and Bone Disorder; Humans; Osteoclasts; Parathyroid Hormone; Tartrates

Vitamin D3 and its hormonally active metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] can be metabolized to a number of daughter metabolites, including 1 alpha,25-(OH)2D3-26,23-lactone; this latter compound has four diastereoisomers. The 23(S),25(R)-lactone (naturally occurring) and the 23(R),25(S)-1 alpha,25-(OH)2D3-26,23-lactone are both known to be able to inhibit bone resorption induced by 1 alpha,25-(OH)2D3 under in vivo or in vitro conditions. To understand the mechanism of the inhibitory action of these two isomers on bone resorption we investigated the effects of 1 alpha,25-(OH)2D3-26,23-lactone on unfractionated mouse bone marrow cells in vitro. The addition of 1 alpha,25-(OH)2D3 to these cultures dose-dependently stimulated the formation of multinucleated cells over a range of 10(-9) - 10(-7) M. The 23(S),25(S)- and 23(R),25(R)-1 alpha,25-(OH)2D3-26,23-lactones also increased the number of multinucleated cells, whereas the 23(S),25(R)- and 23(R),25(S)-1 alpha,25-(OH)2D3-26,23-lactones failed to do so. In addition, these latter two diastereomers inhibited the 1 alpha,25-(OH)2D3 stimulation of multinucleated cell formation, although the 23(S),25(S)- and 23(R),25(R)-1 alpha,25-(OH)2D3-26,23-lactones and 24R,25-(OH)2D3 did not. These multinucleated cells responded to calcitonin and contained tartrate-resistant acid phosphatase, both of which are characteristic of osteoclasts. The present data suggest that inhibition of multinucleated cell formation is the mechanism by which the 23(S),25(R)- or 23(R),25(S)-1 alpha,25-(OH)2D3-26,23-lactone inhibits bone resorption induced by 1 alpha,25-(OH)2D3.

Topics: Acid Phosphatase; Animals; Bone Marrow; Bone Marrow Cells; Bone Resorption; Calcitonin; Calcitriol; Cell Count; Cell Differentiation; Cell Nucleus; Cells, Cultured; Mice; Mice, Inbred ICR; Osteoclasts; Stereoisomerism; Tartrates

Relative activities of acid and alkaline phosphatases in rat reproductive tissue were studied in young adult (10-12 months) and aged (30-34 months) rats. Prostatic alkaline phosphatase alone increased in aged animals. Moreover, prostate preparations contain two isoenzymes of alkaline phosphatase whereas only one was found in epididymal tissues. Age-associated changes in the prostatic alkaline phosphatase isoenzyme ratios were observed. No significant age-related changes in acid phosphatase tartrate sensitivity were found. Techniques for the preparation of prostate and epididymis tissue extracts are reported.

Topics: Acid Phosphatase; Aging; Alkaline Phosphatase; Aluminum; Animals; Creatine Kinase; Epididymis; Glucosephosphate Dehydrogenase; Isoenzymes; Male; Prostate; Rats; Rats, Inbred F344; Rats, Inbred Strains; Tartrates

An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in

Topics: Acid Phosphatase; Animals; Bone Matrix; Cattle; Chromatography; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Isoelectric Focusing; Kinetics; Lysosomes; Molecular Weight; Phosphoprotein Phosphatases; Protein Tyrosine Phosphatases; Substrate Specificity; Tartrates

Aluminum (Al) accumulation in bone is associated with low bone formation and mineralization rates; resorption may also be reduced. The mechanism of these Al-induced changes was investigated using cultured mouse osteoblast-like (OB) and osteoclast-like (OC) cells. The Al effect on bone resorption was measured by the in vitro release of 45Ca and beta-glucuronidase from mouse fetal limb-bones. Al had a biphasic effect. High concentrations (greater than 1.5 X 10(-6) M) of Al inhibited collagen and DNA synthesis, ornithine decarboxylase and alkaline phosphatase activity in OB, and depressed tartrate-resistant acid phosphatase activity in OC. Lower Al concentrations stimulated these cellular activities and 45Ca and beta-glucuronidase release from fetal bones. Al had no effect on basal cAMP levels in OB but inhibited the stimulating effect of bPTH on cAMP content. Al also altered the 1,25(OH)2D3 effects on the ornithine decarboxylase activity of OB cells. These data suggest that: (i) the low bone formation observed in vivo during Al intoxication may be due to the inhibition of collagen synthesis and to depressed cell proliferation; and (ii) Al may indirectly influence bone remodeling by interfering with the actions of bPTH and 1,25(OH)2D3 on bone cells.

Topics: Acid Phosphatase; Alkaline Phosphatase; Aluminum; Animals; Bone and Bones; Bone Resorption; Calcitriol; Cell Division; Cells, Cultured; Collagen; Cyclic AMP; DNA; Mice; Ornithine Decarboxylase; Osteoblasts; Osteoclasts; Parathyroid Hormone; Tartrates

Percoll density gradient centrifugation was used for isolating large quantities of bradyzoites of Sarcocystis suicanis, which were used for enzymatic analysis. Crude extracts of bradyzoites contained activities suggestive of several acid hydrolases. Levels of acid and alkaline phosphatase were higher than those of beta-N-acetylhexosaminidase and beta-galactosidase. Acid phosphatase was purified 156-fold with an overall recovery of 54% using DEAE-Sepharose 4B and Sephadex G-200 chromatography. The partially purified enzyme was not a glycoprotein and had a molecular weight of approximately 170,000. The enzyme was markedly inhibited by Cu++, Hg++, and iodoacetamide, suggesting the presence of a sulfhydryl group. Sodium tartrate caused strong inhibition of the enzyme. The acid phosphatase of S. suicanis appears to be a unique enzyme that cannot be classified under high or low molecular weight acid phosphatases of widely diverse origin.

Topics: Acid Phosphatase; Animals; Copper; Hydrogen-Ion Concentration; Iodoacetamide; Kinetics; Mercuric Chloride; Molecular Weight; Sarcocystis; Substrate Specificity; Tartrates

The immunological similarity between human tartrate-resistant acid phosphatase (EC 3.1.3.2) and porcine uteroferrin previously reported for the isoenzyme from spleens of patients with leukemic reticuloendotheliosis (Ketcham et al., J Biol Chem 1985;260:5768-76) has been confirmed for partly purified acid phosphatase found in the spleen of a patient with Gaucher's disease, and for the corresponding isoenzyme in other tissues and serum. Anti-uteroferrin antibodies raised in rabbits have been used to demonstrate the feasibility of their application in an immunoassay for tartrate-resistant acid phosphatase in serum.

Topics: Acid Phosphatase; Antibodies; Cross Reactions; Humans; Immunodiffusion; Isoenzymes; Metalloproteins; Spleen; Tartrate-Resistant Acid Phosphatase; Tartrates

Cytochemical localization of tartrate-resistant acid phosphatase (TRAP), tartrate-sensitive acid phosphatases (TSAP), alkaline phosphatase, and nonspecific esterase was used to characterize perivascular cells within cartilage canals. In the distal femoral epiphyses of 5- to 7-day-old mice, three stages of canal development can be distinguished, and at each developmental stage different perivascular cells were present with morphological characteristics of degradative cells. Vacuolated cells resembling macrophages, fibroblastic cells, and chondroclasts were present adjacent to the matrix in superficial, intermediate, and deep canals, respectively. In order to characterize these perivascular cells cytochemically, nonspecific esterase and TSAP staining was used to identify macrophages, alkaline phosphatase staining was used to identify fibroblastic cells, and TRAP staining was used to identify chondroclasts. There were no cells present in the canals at any developmental stage that were positive for TSAP or strongly positive for nonspecific esterase, placing doubt on the identity of the vacuolated cells as macrophages. Alkaline phosphatase-positive perivascular cells were present in the intermediate and deep canals adjacent to matrix containing alkaline phosphatase-positive chondrocytes. These alkaline phosphatase-positive cells were found in the same location within canals as the fibroblastic cells. Tartrate-resistant acid phosphatase was localized in chondroclasts at the tips of deep canals but was not confined exclusively to chondroclasts. Except for the very early stage of canal development prior to chondrocyte hypertrophy, TRAP-positive cells were present at the tips of superficial and intermediate canals as well as at the tips of the deep canals.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Carboxylesterase; Carboxylic Ester Hydrolases; Fibroblasts; Growth Plate; Histocytochemistry; Mice; Tartrates

B-cell neoplasias such as CLL can be viewed as models of monoclonal populations restricted within discrete ranges of B-cell maturation. It is unknown whether other B-cell leukemias such as prolymphocytic leukemia (PLL), lymphoplasmacytoid immunocytoma (IC), and hairy cell leukemia (HCL) involve different B lineages or are malignant variants of B cells in successive stages of development along the same lineage. Therefore in vitro maturation was induced with the phorbol ester TPA in leukemic cell samples from 10 CLL, 4 PLL, and 4 IC patients. Morphologically, both plasmacytic and hairy cell-like phenomena were induced. The latter unexpected finding was confirmed by reaction with HD6 (CD22) antibody which stains HCL but is unreactive with plasma cells, multiple myeloma, and CLL cells. Tartrate-resistant acid phosphatase was demonstrated in TPA-cultured CLL, PLL, and IC cells, and the same isoenzyme band as in HCL was revealed by isoelectrofocusing. On the other hand, an increase of IgM messenger RNA was detected in up to 20% of the cells in CLL cultures by single-cell in situ hybridization with fluoro-chrome-labeled DNA probes. An abundance of IgM messenger RNA characterizes lymphoplasmacytoid cells as found in IC. Our data demonstrate that CLL, PLL, and IC can be induced to realize a common genetic program which bears characteristics of HCL indicating that these four entities are more closely related than previously thought.

Topics: Acid Phosphatase; Antigens, Neoplasm; Humans; Immunoglobulin M; Isoenzymes; Leukemia, Hairy Cell; Leukemia, Lymphoid; Lymphoma, Non-Hodgkin; Phenotype; RNA, Messenger; Tartrates; Tetradecanoylphorbol Acetate

An in vitro co-culture system was applied to study the direct and indirect effects of irradiation on osteoclast formation. Osteoclast precursor-free fetal mouse metatarsal bones were employed as osteoclast-forming inductor and periostea dissected from fetal calvaria as source of proliferating progenitor cells. Direct radiation effects on the formation of osteoclasts were assessed in co-cultures of irradiated periostea and non-irradiated bone rudiments. The results showed that the (blood-borne) periosteal progenitors were rather radiosensitive. A radiation 'survival' curve of osteoclast formation in relation to various doses could be constructed yielding a mean lethal dose (Do value) of 0.94 +/- 0.02 Gy and an extrapolation number of 1.67 +/- 0.01. Irradiation of the fetal long bones by low doses, effective for direct elimination of osteoclast progenitor cells, did not indirectly affect osteoclast development from the non-irradiated periosteal progenitor population. However, at relatively high radiation levels, though not lethal for the long bone rudiments, a significant inhibition of osteoclast formation became evident. The results indicate that radiation primarily affects osteoclast formation via a direct action on radiosensitive, proliferating progenitor cells. Injury to long bone models by relatively high radiation doses may also lead to severe disturbance of osteoclast formation kinetics.

Topics: Acid Phosphatase; Animals; Bone and Bones; Cell Differentiation; Cell Survival; Cells, Cultured; Dose-Response Relationship, Radiation; Mice; Osteoclasts; Stem Cells; Tartrates

Tartrate-resistant acid phosphatase (TRAP) has been proposed as a cytochemical marker for osteoclasts. We have developed an improved technique for the localization of TRAP in rat and mouse bone and cartilage. This procedure employs JB-4 plastic as the embedding medium, permits decalcification, and results in improved morphology compared with frozen sections. Peritoneal lavage cells were used to determine the appropriate isomer and concentration of tartrate necessary for inhibition of tartrate-sensitive acid phosphatase. After incubation in medium containing 50 mM L(+)-tartaric acid, osteoclasts and chondroclasts were heavily stained with reaction product. On the basis of their relative sensitivity to tartrate inhibition, three populations of mononuclear cells could also be distinguished. These three populations may represent: heavily stained osteoclast/chondroclast precursors; sparsely stained osteoblast-like cells lining the bone surface; and unstained cells of monocyte-macrophage lineage. Our results are consistent with the use of TRAP as a histochemical marker for study of the osteoclast.

Topics: Acid Phosphatase; Animals; Bone and Bones; Cartilage; Cold Temperature; Decalcification Technique; Histocytochemistry; Isomerism; Methods; Mice; Osteoclasts; Plastics; Rats; Tartrates; Therapeutic Irrigation

Tartrate-resistant acid phosphatase (TRAP) has been used as a cytochemical marker for the cell mediators of bone resorption, osteoclasts and their mononuclear precursors. We have applied a cytochemical method for TRAP to study the dependence of the osteoclast-mediated bone resorption of tooth eruption on the dental follicle, a connective tissue investment of the developing tooth, by analyzing the TRAP activity of mononuclear cells in the dental follicle before and during pre-molar eruption in dogs. The percentage of TRAP-positive monocyte cells increases until mid-eruption, slightly preceding a previously demonstrated rise in numbers of osteoclasts on adjacent bone surfaces. These data suggest an ontogenetic relationship between follicular mononuclear cells and osteoclasts on adjacent alveolar bone surfaces during tooth eruption. However, because TRAP occurs in other tissues and is not an exclusive indicator of pre-osteoclasts, proof of their relationship will have to await application of more definitive techniques.

Topics: Acid Phosphatase; Alveolar Process; Animals; Bone Resorption; Dental Sac; Dogs; Histocytochemistry; Osteoclasts; Tartrates; Tooth Eruption

Suspensions of intact, yeast-like cells of Sporothrix schenckii exhibited an acid phosphatase (EC 3.1.3.2) activity against p-nitrophenyl phosphate of about 5 IU (g dry wt)-1, without recourse to membrane perturbation. This extra-cytoplasmic acid phosphatase was reversibly and competitively inhibited by orthophosphate (Ki = 2 mM at pH 5) but unaffected by L(+)-tartrate (in contradistinction to some of the cytoplasmic acid phosphatases of the same organism). Inactivation by NaF of the extra-cytoplasmic isoenzyme was irreversible and followed first order kinetics; sensitivity to NaF was decreased by the presence of citrate, phosphate or substrate. Neither Km (0.3 mM at pH 5) nor Vmax for this enzyme in acetate buffer was greatly affected by pH in the range 3-5 but the first order rate constant for inactivation by NaF was strongly dependent on pH (maximum at pH 3.5). Crude cell-free extracts of yeast cells had nine electrophoretically distinct acid phosphatase activity bands and, on the basis of the pattern of inhibitors, the extra-cytoplasmic activity was identified as Y-I, an isoenzyme that barely penetrates standard polyacrylamide gel electropherograms. Additional evidence for the assignment came from selective inactivation of this isoenzyme by short treatments of intact cells with NaF under conditions that did not allow penetration of the plasma membrane by the inhibitor and did not kill the cells.

Topics: Acid Phosphatase; Hydrogen-Ion Concentration; Kinetics; Phosphates; Sodium Fluoride; Sporothrix; Tartrates

We improved the spectrophotometric assay of tartrate-resistant acid phosphatase (TrACP; EC 3.1.3.2) activity in serum. During development of the assay we found that human serum contains a dialyzable, mixed-type noncompetitive inhibitor(s) of TrACP activity, the effects of which on the assay were substantially lessened by diluting the serum sample with water before assay and increasing the substrate concentration. Hemolysis releases into serum a significant amount of TrACP activity from erythrocytes, which can be inactivated by incubating the serum at 37 degrees C for 1 h before assay. Our improved assay was reproducible (CV = 5%), and measured within 10% of the amount of added bovine skeletal TrACP activity. Preliminary application of the assay revealed that the amount of serum TrACP activity in patients with skeletal diseases differed from normal values and changed in the same direction as the expected change in bone turnover, suggesting that TrACP activity in serum could be useful clinically as a marker of bone metabolism, possibly of bone resorption.

Topics: Acid Phosphatase; Adult; Aged; Animals; Bone Diseases, Metabolic; Bone Resorption; Cattle; Drug Resistance; Erythrocytes; Female; Humans; Isoenzymes; Male; Middle Aged; Tartrates

Evidence was sought for the presence of osteoclasts or preosteoclasts in cells prepared from neonatal murine calvaria by sequential enzymatic digestion. Freshly isolated cells released late in the digestion process resorbed both living and devitalized calvarial bone matrix in response to PTH, accompanied by the development of osteoclasts. Light and scanning electron microscopy of these cells after 1 to 2 days in culture revealed the presence of round cells (10 to 15 mu in diameter) with minimal surface microvilli in addition to the larger osteoblastic cells. Few cells contained tartrate-resistant acid phosphatase (TRAPase). If initially seeded at confluent density, more cells positive for TRAPase developed during subsequent culture, and these cells retained the ability to resorb calvarial bone matrix upon treatment with parathyroid hormone (PTH). These cultured cells responded to PTH with increased secretion of TRAPase into the medium and increased numbers of TRAPase positive cells. These were 20 to 50 mu in diameter, sometimes multinucleated, and some were spread 100 to 200 mu in length. Observations of living cells that took up neutral red showed that, upon treatment with calcitonin (CT), surface filopodia of some but not all of the labeled cells retracted within 30 minutes. Loss of resorptive response to PTH, as well as PTH-stimulated development of TRAPase-positive cells, occurred if the cells were initially seeded at low density and grown to confluence before exposure to hormone. This correlated with the loss of most of the 10 to 15 mu diameter round cells. These observations suggest that preosteoclasts are present among these small cells that can give rise to osteoclasts upon treatment with PTH.

Topics: Acid Phosphatase; Animals; Animals, Newborn; Bone Resorption; Calcitonin; Cells, Cultured; Cyclic AMP; Mice; Microscopy, Electron, Scanning; Osteoclasts; Parathyroid Hormone; Skull; Tartrates

Cell fractionation of bloodstream Trypanosoma rhodesiense, using isopycnic sucrose gradient centrifugation, reveals acid phosphatase activities against a range of substrates to be associated, to varying degrees, with subcellular particle populations identified as derived from flagella pocket membrane and surface membrane. Using these same substrates (alpha and beta glycerophosphate, p-nitrophenyl phosphate and glucose-6-phosphate) at least two distinct acid phosphatase activities can be distinguished. One is thermolabile (approximately 80% inactivated after 30 min. at 60 degrees C), sensitive to tartrate (50% inhibited at 1.8 mM Na tartrate) with a pH optimum approximately 4.5 and appears to exhibit little substrate preference. The other acid phosphatase is relatively heat stable (approximately 30% inactivated), insensitive to tartrate (greater than 5.0% inhibited using 1.8 mM Na tartrate) exhibits a somewhat higher pH optimum (approximately 6.0) and is more substrate specific (6X more active toward glucose-6-PO4 than beta-glycerophosphate). Further cell fractionation experiments reveal 85% of the tartrate sensitive acid phosphatase to be associated with flagella pocket membrane and to account for 80% of the organisms hydrolytic activity toward beta-glycerophosphate. The tartrate resistant acid phosphatase however, has a much less exclusive localization being almost equally distributed between surface membrane (40%) and flagella pocket membrane (60%).

Topics: Acid Phosphatase; Animals; Cell Membrane; Flagella; Hot Temperature; Hydrogen-Ion Concentration; Pyrophosphatases; Subcellular Fractions; Substrate Specificity; Tartrates; Trypanosoma brucei brucei

Tartrate-resistant acid phosphatase active on nucleoside di- and triphosphate substrates was isolated from developing rat bone and purified 2500-fold. The enzyme concentration had a purple coloration and activity that was sensitive to reducing agents. Mild reducing agents such as ferrous ion and ascorbic acid caused loss of purple color and increased activity toward substrates severalfold; however, a strong reductant such as dithionite caused loss of both color and activity which were partially restored by addition of ferrous ion and ascorbic acid. Enzyme activity was homogeneous with protein during the final gel permeation steps of chromatography and gave an apparent molecular size of about 40,000 Da. Determination of iron in the most pure preparation revealed the presence of 1.3 atoms of iron per molecule of the tartrate-resistant enzyme E2. Other properties of the purified enzyme include a pI of approximately 9.5 and sensitivity to inhibition by ions of copper, zinc, fluoride, and molybdate. Antibody prepared to the pre-concanavalin A (Con A)-Sepharose purified enzyme reacted with all protein from the Con A step, but it did not react with tartrate-sensitive acid phosphatase from rat bone or with potato acid phosphatase. Purple acid phosphatase from rat bone has many properties that parallel the iron-containing purple acid phosphatases from rat spleen, bovine spleen, and pig uterine secretions.

Topics: Acid Phosphatase; Animals; Bone and Bones; Bone Development; Chromatography, Affinity; Electrophoresis, Disc; Enzyme Activation; Immunodiffusion; Isoelectric Focusing; Protein Denaturation; Rats; Rats, Inbred Strains; Substrate Specificity; Tartrates

Topics: Acid Phosphatase; Adolescent; Adult; Bone Resorption; Female; Humans; Hyperparathyroidism; Male; Middle Aged; Ovariectomy; Tartrates

Tartrate-resistant acid phosphatase (TRAcP) is used as a marker for osteoclasts, which are believed to be derived from phagocytic cells or phagocyte stem cell precursors. To further investigate the relationship between monocytic phagocytes and osteoclasts, acid phosphatase (AcP) activity was measured by three different techniques in human peripheral blood monocytes, monocyte-derived macrophages, and the U937 cell line. We found that cytochemistry and gel electrophoresis led to similar results, but that the colorimetric assay was inconsistent. Normal human peripheral monocytes expressed both tartrate-sensitive and -resistant AcP. In culture these cells formed polykaryons and expressed TRAcP activity that was further identified as an isoenzyme associated with bone tissue. In contrast, the U937 cells did not express TRAcP activity as measured by gel electrophoresis. Both U937 cells and monocytes possess material that interferes with interpretation of the colorimetric assay of AcP. The presence of TRAcP in monocyte-derived macrophages further supports the relationship between phagocytic cells and bone osteoclasts.

Topics: Acid Phosphatase; Calcitriol; Cell Line; Colorimetry; Histocytochemistry; Humans; Isoenzymes; Lymphoma, Large B-Cell, Diffuse; Macrophages; Monocytes; Tartrates

Forty isolates of Leishmania, representing all major species infecting humans and one parasite of lizards, were examined for their ability to secrete an extracellular acid phosphatase activity. This enzyme, which was originally described and characterized from a Sudanese strain of L. donovani, was detected in the culture supernatants of all species of promastigotes examined except for L. major and L. tarentolae. There were quantitative differences among species in their levels of enzyme activity and in the sensitivity of the exoenzyme to inhibition by L(+) tartrate. Upon electrophoresis in nondenaturing polyacrylamide gels, extracellular acid phosphatase from L. braziliensis panamensis, L. tropica, and L. mexicana showed distinctive patterns which were similar for all isolates of a given species, while enzymes from L. donovani isolates differed from one another in relative electrophoretic mobility. Enzymes from all species appeared heterogeneous, showing either discrete multiple bands or single diffuse bands on gels stained for enzyme activity. Although the biological function of the extracellular acid phosphatase is presently unknown, the exoenzyme may be of value as a diagnostic or taxonomic characteristic.

Topics: Acid Phosphatase; Animals; Electrophoresis, Polyacrylamide Gel; Humans; Leishmania; Leishmania donovani; Leishmania tropica; Lizards; Tartrates

Positive identification of osteoclast percursors has not yet been possible. The authors have, in the present report, used a model system in the rat in which it is possible to induce the formation of multinucleated osteoclasts at a predictable and reproducible site and time (Tran Van P, Vignery A, Baron R. Anat Rec 1982, 202:445-451; Cell Tissue Res 1982, 225:283-292). This system allowed the investigation of the cellular events occurring locally during the recruitment and differentiation of osteoclast precursors. Prior to the formation of multinucleated osteoclasts, mononuclear cells positive for fluoride-inhibitable nonspecific esterase and cells positive for tartrate-resistant acid phosphatase increase in number locally. Double staining procedures demonstrated the presence of both enzymes in a number of cells, thereby suggesting that they are steps in the differentiation of a single cell population. Ultrastructural studies show that lysosomal enzymes are present in every compartment of the biosynthetic pathway, in small primary lysosomes and various forms of storage granules. As these precursors arrive at the bone surface, the storage granule lysosomes are markedly depleted. It is concluded that mononuclear precursors of the osteoclast are members of the mononuclear-phagocyte lineage and differentiate early to synthesize, store, and later secrete large quantities of lysosomal enzymes. The mature osteoclast, which, as its precursor, is positive for the mononuclear-phagocyte marker enzyme nonspecific esterase, results from the fusion of these mononuclear precursors, which occurs only after their attachment to the bone surface to be resorbed.

Topics: Acid Phosphatase; Animals; Bone Resorption; Carboxylesterase; Carboxylic Ester Hydrolases; Cell Differentiation; Cell Nucleus; Cytoplasm; Fluorides; Histocytochemistry; Kinetics; Lysosomes; Macrophages; Male; Microscopy, Electron; Osteoclasts; Periosteum; Phagocytes; Rats; Rats, Inbred Strains; Stem Cells; Tartrates

Topics: Acid Phosphatase; Carcinoma, Basal Cell; Clinical Enzyme Tests; Diagnosis, Differential; Fibrosarcoma; Histiocytoma, Benign Fibrous; Humans; Liposarcoma; Rhabdomyosarcoma; Soft Tissue Neoplasms; Tartrates

This report describes the qualitative acid phosphatase (acP) isoenzyme profiles detected in permanent human hematopoietic cell lines. The acP activity was separated into its isoenzymes by isoelectric focusing on horizontal thin-layer polyacrylamide gels. The pattern of acP isoenzyme was investigated in a total of 86 cell lines. These cell lines were classified into five groups on the basis of their phenotypes characterized in the multiple marker analysis: 74 leukemia-lymphoma cell lines (26 T-, 34 B-, 6 myelomonocytic, 8 Non-T, Non-B cell lines) and 12 so-called 'normal' Epstein-Barr virus transformed B-lymphoblastoid cell lines. Their immunological features had been analysed in detail by use of a large panel of poly- and monoclonal antibodies which led to a further subclassification into stages of differentiation. A progressive increase in number and staining intensity of the isoenzymes which paralleled the expression of surface markers at different stages of differentiation along their developmental pathway was seen in the T- and B-leukemia-lymphoma cell lines. Some cell lines whose isoenzyme profiles did not correspond to the stage of differentiation as evidenced by surface antigen analysis might represent good examples of deranged gene expression in otherwise normally programmed malignant cells, i.e. in our study a mismatch between the isoenzymatic and immunological phenotypes. The tartrate-resistant isoenzyme was detected in 9 out of 74 leukemia-lymphoma cell lines (4 T-, 2 B-, 1 myelomonocytic, 2 Non-T, Non-B cell lines) and in 10 out of 12 normal B-lymphoblastoid cell lines; the only one studied hairy cell leukemia cell line did not express this isoenzyme. The relative specificity of the tartrate-resistant acP is discussed in detail. No leukemia-lymphoma specific isoenzyme or an additional isoenzyme which was not seen in normal hematopoietic cells could be observed. Nor did we find an isoenzyme or isoenzyme pattern characteristic for a certain cell lineage. This underlines the necessity of a combined analysis using markers from different disciplines in the 'multiple marker analysis' in order to accurately characterize normal and malignant blood cells. Furthermore, our results support the concept of maturation arrest at particular stages of differentiation together with the theory of normal gene expression in leukemic cells equivalent to that in their normal counterparts.

Topics: Acid Phosphatase; Antigens, Surface; B-Lymphocytes; Cell Differentiation; Cell Line; Humans; Isoenzymes; Leukemia; Leukemia, Lymphoid; Leukemia, Monocytic, Acute; Leukemia, Myeloid, Acute; Lymphoma; Phenotype; T-Lymphocytes; Tartrates

Extracts of human lung tissue contain appreciable activities of a tartrate-resistant acid phosphatase which is apparently identical with the analogous enzyme in bone extracts, with respect to electrophoretic mobility, apparent molecular weight (ca. 37,000), Michaelis constants and relative rates of hydrolysis of various substrates. The acid phosphatase appears to be a constituent of alveolar macrophages. Lung provides a convenient source for the preparation of tartrate-resistant acid phosphatase.

Topics: Acid Phosphatase; Bone and Bones; Chromatography, Gel; Chromatography, Ion Exchange; Drug Resistance; Humans; Lung; Tartrates

We examined cellular relationships and cytokinetics of hairy cells in colonies formed in an in vitro cloning system. Cells in small colonies and at the periphery of larger ones were separated by wide, irregular intercellular spaces. Cells in the core of large colonies were elaborately intertwined by their cytoplasmic processes and so densely packed that their intercellularity was greatly reduced. Cell labeling with 3H-thymidine revealed indices ranging from less than 1% to 35%. The combination of autoradiography with a stain for tartrate-resistant acid phosphatase showed that the enzyme was not expressed by cells in S-phase. The cellular relationships of hairy cells in colonies grown in this system closely mimic those of their in vivo counterparts in the spleen and bone marrow.

Topics: Acid Phosphatase; Autoradiography; Cell Communication; Cell Division; Cell Separation; Clone Cells; Culture Media; Humans; Kinetics; Leukemia, Hairy Cell; Microscopy, Electron; Microvilli; Pseudopodia; Tartrates

The immunological cross-reactivity of heterogeneous acid phosphatase isozymes from different human tissues has been studied using monospecific antisera prepared against four homogeneous acid phosphatases. The enzyme characterized as tartrate-inhibitable, prostatic acid phosphatase is also found to be present in leukocytes, kidney, spleen, and placenta. The tartrate-inhibitable (liver) lysosomal enzyme is also found in kidney, fibroblasts, brain, placenta, and spleen, but it is not detectable in erythrocytes and prostate. In several tissues, 10-20% of the tartrate-inhibitable enzyme is not precipitated by any of the antisera used; an exceptionally high amount (54%) of such an enzyme is present in human brain. Antiserum against a low molecular weight tartrate-resistant liver enzyme (14 kDa) does not crossreact with the erythrocyte enzyme. (10-20 kDa). All other tissues except placenta, prostate, and fibroblast cells show a cross-reactivity with the 14-kDa acid phosphatase antiserum. Thus, the low molecular weight human liver acid phosphatase is distinct from the erythrocyte enzyme, and there are also at least three different tartrate-inhibitable acid phosphatases in human tissues. Chromosomal assignments have been made for only two of the (at least) five acid phosphatases that are present in adult human tissues.

Topics: Acid Phosphatase; Blood Cells; Brain; Cross Reactions; Female; Humans; Isoenzymes; Kidney; Liver; Male; Placenta; Prostate; Spleen; Tartrates

Tartrate-inhibitable acid phosphatase was purified to apparent homogeneity from human placenta. The enzyme is composed of two subunits with an apparent molecular mass of 48 kDa. Each subunit carries one oligosaccharide of the high-mannose/hybride type. The purified enzyme has an isoelectric point of pH 6.2. It cleaves phosphomonoester bonds at acid pH, is competitively inhibited by L-tartrate, Ki = 0.51 microM, and phosphate, Ki = 0.8mM. A monospecific antiserum raised against the purified placental enzyme precipitated 62% and 85% of the tartrate-inhibitable acid phosphatase present in extracts of placenta and fibroblasts, respectively. By means of subcellular fractionation and immunoprecipitation it was shown that the majority of tartrate-inhibitable acid phosphatase is located in lysosomes in normal and mucolipidosis II fibroblasts. In the human Hep G-2 hepatoma cells a significant fraction of the enzyme appears to be associated with non-lysosomal organelles.

Topics: Acid Phosphatase; Carcinoma, Hepatocellular; Cell Line; Electrophoresis, Polyacrylamide Gel; Female; Fibroblasts; Humans; Hydrolysis; Isoelectric Focusing; Liver Neoplasms; Placenta; Pregnancy; Subcellular Fractions; Tartrates

In 46 patients with primary hyperparathyroidism, in 21 non-dialysed patients with advanced renal failure, and in 52 patients on hemodialysis, a significant positive correlation was found between bone isoenzyme of serum alkaline phosphatase and plasma tartrate resistant acid phosphatase. In primary hyperparathyroidism, a significant positive correlation was found between the radiological degree of osteodystrophy and the biochemical parameters of bone remodelling. After removal of the parathyroid adenoma, only the tartrate-resistant acid phosphatase decreased to normal limits. Plasma tartrate resistant acid phosphatase was most significantly influenced by serum immunoreactive parathyroid hormone levels. In chronic renal failure, bone isoenzyme of serum alkaline phosphatase was most significantly influenced by serum immunoreactive parathyroid hormone levels, by hypocalcemia and by duration of hemodialysis. The results confirm that in hyperparathyroidism the extent of the whole-body rates of bone resorption and formation are approximately equal. The biochemical parameters can be used for serial assessment of the course of the disease but are not specific for diagnosis.

Topics: Acid Phosphatase; Alkaline Phosphatase; Bone and Bones; Humans; Hyperparathyroidism; Isoenzymes; Kidney Failure, Chronic; Renal Dialysis; Tartrates

Acid phosphatases of the rat ventral prostate were fractionated by gel filtration (GF) on Sepharose 6B, isoelectric focusing (IEF), and chromatofocusing (CF). In GF three activity peaks (GF-1, GF-2, GF-3) were disclosed. They showed some differences in substrate preference when six substrates (p-nitrophenyl phosphate; p-NPP; phenolphthalein phosphate, Phe-P; thymolphthalein phosphate, Tym-P; alpha-naphthyl phosphate, alpha-NP; beta-naphthyl phosphate, beta-NP; naphthol ASBI phosphate, N-ASBI-P) were tested. Differences were also encountered in their sensitivity to tartrate and fluoride. IEF gave seven bands at different pI values (8.3, 8.1, 7.9, 7.1, 6.4, 5.5, and 5.0) with alpha-NP and beta-NP but only four with N-ASBI-P. Four of the bands (8.3, 8.1, 7.9, 5.5) were sensitive to tartrate. In CF eight activity peaks (CF-1 to CF-8) were resolved with the six substrates. They differed from each other in pI values, pH optima, substrate preference, and modifier characteristics. Peaks CF-1 (pI 8.3, pH 5.5), CF-2 (pI 8.1, pH 4.2) and CF-3 (pI 7.9, pH 4.2) had a large substrate spectrum and high sensitivity to tartrate and fluoride. CF-4 (pI 7.1, pH 6.0) and CF-7 (pI 5.5, pH 4.2) were low in activity, preferred alpha-NP as substrate, and were moderately sensitive to tartrate. CF-5 (pI 6.4, pH 5.5) and CF-8 (pI 5.0, pH 5.0) were able to hydrolyse all substrates tested with moderate inhibition by tartrate. CF-6 (pI 6.0, pH 5.0) showed a relative preference for p-NPP and Phe-P with no hydrolysis of N-ASBI-P and Tym-P. Of these activities CF-6 and CF-7 were also clearly activated by Co2+. Peaks CF-6 and CF-7 appeared the most sensitive to p-chloromercuribenzoate. It is concluded that activities CF-1, CF-2, and CF-3 are lysosomal isoenzymes with minor structural differences. The others are possibly all nonlysosomal with greater biochemical differences. Some of them apparently represent the secretory form(s) of acid phosphatase in the rat ventral prostate.

Topics: Acid Phosphatase; Animals; Chromatography; Chromatography, Gel; Fluorides; Isoelectric Focusing; Isoelectric Point; Isoenzymes; Male; Prostate; Rats; Rats, Inbred Strains; Substrate Specificity; Tartrates

The staining intensity and inhibitor sensitivity of acid phosphatase activity was determined histochemically in various tissues of normal and ia rat pups by the use of freeze-dried whole body sections. Activity was determined using alpha-naphthylphosphate as substrate and hexazonium pararosaniline as coupler. Sections from ia rats (6 and 24 days old) showed markedly higher enzyme activity in bone than sections from normal littermates. However, there were no differences between ia and normal pups in acid phosphatase activity in soft tissues and developing teeth. Preincubation of sections with 1-100 mM sodium dithionite (an iron-binding agent) caused a dose-related inhibition of enzyme activity in bone of ia and normal pups, but only slight inhibition of activity in soft tissues. Partial restoration of the dithionite-inhibited activity in bone was achieved by subsequent preincubation in 1 mM FeCl2. Addition of 100 mM sodium tartrate to the staining solution of non-preincubated sections caused almost complete inhibition of activity in soft tissues and the developing teeth but no inhibition of the activity in bone that was sensitive to sodium dithionite. These data indicate a) that sodium dithionite can be used as a specific histochemical inhibitor of the tartrate-resistant acid phosphatase and b) that the source of increased acid phosphatase activity in bone from ia rats is mostly from the tartrate-resistant acid phosphatase.

Topics: Acid Phosphatase; Animals; Bone and Bones; Brain; Dithionite; Ferrous Compounds; Ganglia, Spinal; Muscles; Osteopetrosis; Rats; Rats, Mutant Strains; Tartrates; Tooth Abnormalities

Topics: Acid Phosphatase; Adult; Bone Diseases; Child; Child, Preschool; Colorimetry; Electrophoresis, Polyacrylamide Gel; Gaucher Disease; Humans; Immunoenzyme Techniques; Infant; Middle Aged; Splenectomy; Tartrates

Topics: Acid Phosphatase; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Colorimetry; Dengue; Electrophoresis; Humans; Immunoenzyme Techniques; Molecular Weight; Substrate Specificity; Tartrates

Acid phosphatase (AcP) in neoplastic cells from various lymphoid leukemias was examined. In the cytochemical studies, tartrate-resistant AcP (T-rAcP) activity was observed in the neoplastic cells from well-differentiated lymphoid leukemias such as adult T-cell leukemia (ATL), B-cell chronic lymphocytic leukemia (B-CLL), T-cell chronic lymphocytic leukemia (T-CLL), and hairy-cell leukemia (HCL). T-rAcP activity was also detected in a small number of leukemic cells obtained from T-cell acute lymphoblastic leukemia (T-ALL), while it was not detected in the neoplastic cells from null-ALL, macroglobulinemia, and multiple myeloma (MM). In the electrophoretical studies, fraction 1 (F-1), F-3, F-3b, and F-4 were completely tartrate-sensitive, while F-2 was partially resistant and F-5 was completely resistant. T-rAcP activity (F-5) was observed in ATL cells, B-CLL cells, and HCL cells, while it was not detected in ALL cells, macroglobulinemia cells, and MM cells. The present study indicates that T-rAcP activity is observed not only in HCL cells but also in the well-differentiated lymphoid cells such as ATL cells, B-CLL and T-CLL cells except the most highly differentiated forms of B-cells of MM and macroglobulinemia.

Topics: Acid Phosphatase; Cell Transformation, Neoplastic; Electrophoresis, Polyacrylamide Gel; Humans; Isoenzymes; Leukemia, Hairy Cell; Leukemia, Lymphoid; Multiple Myeloma; T-Lymphocytes; Tartrates; Waldenstrom Macroglobulinemia

Topics: Acid Phosphatase; Adult; Humans; Isoenzymes; Leukemia; Male; Tartrates

Topics: Acid Phosphatase; Enzyme Inhibitors; Humans; Male; Neoplasms; Prostatic Hyperplasia; Prostatic Neoplasms; Tartrates

Topics: Acid Phosphatase; Bone Neoplasms; Clinical Enzyme Tests; Diagnosis, Differential; Diethylstilbestrol; Drug Therapy; Enzyme Inhibitors; Geriatrics; Humans; Male; Neoplasm Metastasis; Neoplasms; Osteosclerosis; Pathology; Prostatectomy; Prostatic Hyperplasia; Prostatic Neoplasms; Radiography; Tartrates

Topics: Acid Phosphatase; Animals; Phosphoric Monoester Hydrolases; Rats; Tartrates

Topics: Acid Phosphatase; Humans; Phosphoric Monoester Hydrolases; Phylogeny; Succinates; Tartrates

Topics: Acid Phosphatase; Blood; Humans; Male; Phosphoric Monoester Hydrolases; Prostatic Neoplasms; Protein Tyrosine Phosphatases; Tartrates

Topics: Acid Phosphatase; Blood; Humans; Male; Phosphoric Monoester Hydrolases; Prostate; Protein Tyrosine Phosphatases; Tartrates

Topics: Acid Phosphatase; Acids; Blood; Humans; Hypertrophy; Male; Phosphoric Monoester Hydrolases; Prostatic Hyperplasia; Prostatic Neoplasms; Protein Tyrosine Phosphatases; Tartrates

Topics: Acid Phosphatase; Humans; Tartrates