4-aminophenylmercuriacetate and 1-10-phenanthroline

We previously identified angiotensin converting enzyme (ACE) and an endopeptidase activity that degraded angiotensin-(1-7) [Ang-(1-7)] to Ang-(1-5) and Ang-(1-4), respectively, in the cerebrospinal fluid (CSF) of 6-month old male sheep. The present study undertook a more comprehensive analysis of the CSF peptidase that converts Ang-(1-7) to Ang-(1-4) in control and in utero betamethasone-exposed sheep (BMX). Characterization of the Ang-(1-7) peptidase revealed that the thiol agents 4-aminophenylmercuric acetate (APMA) and p-chloromercuribenzoic acid (PCMB), as well as the metallo-chelators o-phenanthroline and EDTA essentially abolished the enzyme activity. Additional inhibitors for serine, aspartyl, and cysteine proteases, as well as selective inhibitors against the endopeptidases neprilysin, neurolysin, prolyl and thimet oligopeptidases did not attenuate enzymatic activity. Competition studies against the peptidase revealed similar IC50s for Ang-(1-7) (5μM) and Ang II (3μM), but lower values for Ala(1)-Ang-(1-7) and Ang-(2-7) of 1.8 and 2.0μM, respectively. In contrast, bradykinin exhibited a 6-fold higher IC50 (32μM) than Ang-(1-7) while neurotensin was a poor competitor. Mean arterial pressure (78±1 vs. 94±2mmHg, N=4-5, P<0.01) and Ang-(1-7) peptidase activity (14.2±1 vs 32±1.5fmol/min/ml CSF, N=5, P<0.01) were higher in the BMX group, and enzyme activity inversely correlated with Ang-(1-7) content in CSF. Lower Ang-(1-7) expression in brain is linked to baroreflex impairment in hypertension and aging, thus, increased activity of an Ang-(1-7) peptidase may contribute to lower CSF Ang-(1-7) levels, elevated blood pressure and impaired reflex function in this model of fetal programming.

Topics: Aging; Angiotensin I; Animals; Baroreflex; Edetic Acid; Hypertension; Male; p-Chloromercuribenzoic Acid; Peptide Fragments; Peptidyl-Dipeptidase A; Phenanthrolines; Phenylmercuric Acetate; Sheep; Substrate Specificity

Endothelial cells (ECs) are subjected to oxidative stress during many pathological processes, including ischemia/reperfusion and general inflammation. In the present study, we examined the effects of oxidative stress on rates of apoptosis in EC cultures. We treated large and microvessel ECs with menadione for 1 h in vitro to simulate the most common physiological form of oxidative stress, exposure to O2*-. Capillary ECs were resistant to menadione-induced apoptosis when compared with large-vessel ECs. Treatment with 35 microM menadione resulted in an apoptotic rate of approximately 5% in capillary EC cultures compared with approximately 45% in large-vessel EC cultures. At higher concentrations of menadione (35-75 microM), both types of ECs exhibited a concentration-related increase in apoptosis. Necrotic cell death only became evident at menadione concentrations ranging from 75-100 microM for both cell types. The timing of the apoptotic response to a 1 h menadione exposure was very specific. For both EC types, peaks of apoptosis occurred in two distinct waves, at 6-8 and 18-22 h after treatment. Analysis of the events leading up to the first peak of apoptosis indicated that specific matrix metalloproteinases (MMPs) were activated, suggesting that MMPs may be involved in initiating the apoptotic process.

Topics: Animals; Apoptosis; Cattle; Cells, Cultured; Culture Media, Conditioned; Dose-Response Relationship, Drug; Endothelium, Vascular; Glutathione; Matrix Metalloproteinases; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Oxidative Stress; Phenanthrolines; Phenylmercuric Acetate; Superoxides; Time Factors; Vitamin K

Members of the matrix metalloproteinase (MMP) family are responsible for breakdown of extracellular matrix components involved in morphogenetic remodeling of animal embryogenesis. The highly sensitive assay of MMP using synthetic fluorescence-quenching substrate was employed to detect and to characterize a veiled MMP activity expressed in Japanese flounder embryos undergoing formation of lenses. The MMP activity was enhanced in proportion to increasing protein amounts of the embryonic lysate over 5 microg, and this reaction was proceeded in a time-dependent manner and with increasing substrate concentrations. Almost 2-fold increase in the embryonic MMP activity occurred by treatment with 4-aminophenylmercuric acetate, but the activity was markedly suppressed by metal chelating reagents. These enzymatic characteristics are apparently consistent with those of mammalian embryonic MMPs, particularly MMP-9. The characterized MMP activity was highly expressed at the specificstage during embryogenesis, indicating that this MMP may be involved in formation of lenses.

Topics: Animals; Edetic Acid; Enzyme Activation; Enzyme Inhibitors; Flounder; Fluorescent Dyes; Fluorometry; Lens, Crystalline; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Peptides; Phenanthrolines; Phenylmercuric Acetate

Novel gelatinolytic activities in both latent and active forms were detected in the normal organs of rat by gelatin zymography. Multiple active bands were detected in the extracts from the skin, jejunum, muscle, and kidney without any activation. These activities were inhibited by 1,10-phenanthroline or leupeptin, nor by E64, suggesting that these activities were derived from metallo-proteinases or serine-proteinases. Some gelatinolytic active bands were newly induced or enhanced by p-aminophenylmercuric acetate. These results suggest that matrix degrading activities due to metallo- and serine-proteinases were constitutively expressed in various rat normal organs.

Topics: Animals; Cattle; Enzyme Inhibitors; Gelatin; Leupeptins; Male; Metalloendopeptidases; Phenanthrolines; Phenylmercuric Acetate; Rats; Rats, Wistar; Serine Endopeptidases

Remodeling of extracellular matrix (ECM) is one of the key events in many developmental processes. In the present study, a temporal profile of gelatinase activities in regenerating salamander limbs was examined zymographically. In addition, the effect of retinoic acid (RA) on these enzyme activities was examined to relate the pattern-duplicating effect of RA in limb regenerates with gelatinase activities. During regeneration, various types of gelatinase activities were detected, and these activities were at their maximum levels at the dedifferentiation stage. Upon treatment with chelating agents EDTA and 1,10-phenanthroline, the enzyme activities were inhibited indicating that those enzymes are likely matrix metalloproteinases (MMPs). Considering the molecular sizes and the decrease of molecular sizes by treatment with p-aminophenylmercuric acetate, an artificial activator of proMMP, some of the gelatinases expressed during limb regeneration are presumed to be MMP-2 and MMP-9. In RA-treated regenerates, overall gelatinase activities increased, especially the MMP-2-like gelatinase activity which increased markedly. These results suggest that MMP-2-like and MMP-9-like gelatinases play a role in ECM remodeling during regeneration, and that gelatinases are involved in the excessive dedifferentiation after RA treatment.

Topics: Ambystoma mexicanum; Animals; Cell Differentiation; Chelating Agents; Edetic Acid; Enzyme Activation; Extremities; Gelatinases; Phenanthrolines; Phenylmercuric Acetate; Regeneration; Time Factors; Tissue Inhibitor of Metalloproteinases; Tretinoin; Urodela

Evidence suggests that thrombospondin-1 (TSP-1), a 450-kDa glycoprotein in platelets and extracellular matrix, is involved in angiogenesis. However, the mechanisms by which TSP-1 regulates angiogenesis are unknown, and the exact role of TSP-1 in angiogenesis has been controversial: both stimulatory and inhibitory effects of TSP-1 have been reported. In this study, we evaluated the effect of TSP-1 on the capacity of bovine aortic endothelial (BAE) cells to both invade and form microvessel-like tubes in collagen gels. BAE cell tube formation was enhanced by exogenous TSP-1 at relatively low concentrations (1-10 microg/ml) but inhibited at higher concentrations of TSP-1 (>15 microg/ml). In addition, we correlated this biphasic effect on tube formation with the capacity of TSP-1 to stimulate the activity of a matrix metalloproteinase-9 (MMP-9) in BAE cell collagen gel cultures. The TSP-1-mediated stimulation of MMP-9 activity was specific and dose- and time-dependent. Furthermore, TSP-1-stimulated BAE cell invasion and tube formation were reversed by antibodies against both TSP-1 and MMP-9, suggesting that TSP-1 modulates endothelial cell invasion and morphogenesis in vitro by a mechanism involving the regulation of MMP-9 activity. These findings support the conclusion that TSP-1 is a multifunctional modulator of angiogenesis and are consistent with the dynamic presence of TSP-1 in remodeling tissues in which matrix degradation is required.

Topics: Animals; Cattle; Cell Division; Cell Movement; Cells, Cultured; Collagen; Collagenases; Culture Media, Conditioned; Endothelium, Vascular; Extracellular Matrix Proteins; Fibronectins; Gelatinases; Gels; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Membrane Glycoproteins; Neovascularization, Physiologic; Oligopeptides; Phenanthrolines; Phenylmercuric Acetate; Protease Inhibitors; Thrombospondins; Up-Regulation

Matrix metalloproteinases (MMPs) can be activated in vitro by multiple mechanisms such as treatment with proteases, organomercurials, oxidants, and detergents. The proposed cysteine switch model for activation suggests that these multiple methods for activation cause the dissociation of the single cysteine residue in the propeptide from the active site zinc. In particular, it has been suggested that organomercurials such as 4-aminophenylmercuric acetate (APMA) work by directly reacting with the sulfhydryl group of this cysteine residue, resulting in its displacement from the active site. However, recent data by Chen et al. [(1993) Biochemistry 32, 10289-10295] demonstrated that modification of this cysteine residue in the propeptide of stromelysin-1 by sulfhydryl reagents did not result in an active enzyme as predicted. To investigate the roles that this cysteine residue and the propeptide salt bridge (R74 to D79) might play in the APMA-induced activation of stromelysin-1, we have changed these residues by site-directed mutagenesis. Wild-type stromelysin-1 and the mutants were all expressed at detectable levels using a recombinant vaccinia virus system and determined to be catalytically competent by zymography. The wild-type stromelysin-1 and the cysteine mutants (C75S and C75H) underwent APMA-induced activation as determined by the characteristic reduction in molecular weight associated with activation and by their ability to cleave casein only when activated. On the other hand, mutants R74K, D79A, and C75H/D79A did not undergo APMA-induced activation. These results demonstrate that APMA-induced activation of stromelysin-1 involves protein interactions in addition to those with cysteine-75 in the propeptide and also suggest that the R74 to D79 salt bridge may play a role.

Topics: Amino Acid Sequence; Base Sequence; Binding Sites; Blotting, Western; Caseins; Chromatography, Affinity; Cysteine; DNA Primers; Enzyme Activation; Humans; Matrix Metalloproteinase 3; Metalloendopeptidases; Molecular Sequence Data; Mutagenesis, Site-Directed; Phenanthrolines; Phenylmercuric Acetate; Recombinant Proteins; Trypsin; Tumor Cells, Cultured; Vaccinia virus; Zinc

Human umbilical vein endothelial cells (HUVECs) were used to gain insight into the molecular mechanism whereby the major extracellular protease from group A streptococci damages host tissue. HUVECs exposed to streptococcal cysteine protease (SCP) for various times exhibited cytopathic effect and cell detachment from the culture vessel. Gelatin substrate zymography showed that a time- and concentration-dependent increase in the level of activity of an approximately 66-kDa gelatinase occurred in culture medium taken from cells exposed to enzymatically active SCP. This gelatinase comigrated in gelatin zymograms with the activated form of purified recombinant matrix metalloprotease 2 (MMP-2) and had type IV collagenase activity. In contrast, medium taken from cells exposed to inactivated (boiled) SCP and cells exposed to SCP inhibited by treatment with N-benzyloxycarbonyl-leucyl-valyl-glycine diazomethyl ketone lacked the 66-kDa gelatinase. Appearance of the 66-kDa gelatinase activity was also prevented by 1,10-phenanthroline, a zinc chelator and MMP inhibitor. Inasmuch as proteolytically active SCP is required for the emergence of this gelatinase and MMP activation occurs by proteolytic processing, the 66-kDa gelatinase may be a proteolytic cleavage product of a latent MMP expressed extracellularly by HUVECs. Direct SCP treatment of culture supernatant taken from HUVECs not exposed to SCP also produced the 66-kDa gelatinase. The data show that SCP activates an MMP produced by human endothelial cells, a process that may contribute to endothelial cell damage, tissue destruction, and hemodynamic derangement observed in some patients with severe, invasive group A streptococcal infection.

Topics: Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Endothelium, Vascular; Enzyme Activation; Extracellular Matrix; Fibronectins; Gelatinases; Humans; Matrix Metalloproteinase 2; Metalloendopeptidases; Oligopeptides; Phenanthrolines; Phenylmercuric Acetate

Little is known about matrix metalloproteinases (MMPs), enzymes that are required for the structural remodeling and angiogenesis that occur in the corpus luteum (CL) during the first several days postovulation. In fact, little attention has focused on early CL function including the regulation of progesterone (P4) production. Thus, the objective of the present study was 1) to investigate the effects of insulin, LH, and dibutyryl cAMP on P4 production and cell numbers and 2) to identify MMPs in the 4-day-old CL, with use of a defined culture system. Cultures were seeded with either 1 x 10(6) or 0.5 x 10(6) cells. All cultures containing insulin had higher P4 levels and cell numbers (p < 0.05) than those without. In cultures containing insulin, basal P4 levels were high throughout the culture period. Furthermore, neither LH nor dibutyryl cAMP stimulated P4 production (p > 0.05) at a seeding density of 1 x 10(6), whereas they stimulated P4 production (p < 0.05) at a seeding density of 0.5 x 10(6) on Days 6 and 8 of culture. In conditioned medium of control cultures seeded with 0.5 x 10(6) cells, substrate gel electrophoresis (zymography) showed two intense bands that migrated at M(r) approximately 97,000 and approximately 65,000-64,000, while two weaker ones migrated at M(r) approximately 88,000 and approximately 64,000-63,000. The molecular weights of the M(r) approximately 97,000 and approximately 88,000 species were consistent with MMP-9 family members, while the molecular weights of the M(r) approximately 65,000-64,000 and approximately 64,000-63,000 species were consistent with MMP-2 family members.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bucladesine; Cattle; Cell Count; Cells, Cultured; Corpus Luteum; Culture Media, Conditioned; Estrus; Female; Gelatinases; Insulin; Luteinizing Hormone; Metalloendopeptidases; Molecular Weight; Phenanthrolines; Phenylmercuric Acetate; Progesterone; Protease Inhibitors; Time Factors

Significant amounts of collagenase and caseinase activity were detected in infected synovial fluid samples. Partial characterisation of the enzymes by gel filtration suggested that synovial fluid from cases of infectious arthritis may contain enzymes from both the synovial cells and neutrophils. This finding was also supported by analysis of sequential synovial fluid samples from 4 infected joints. In 3 joints the concentration of caseinase and in 1 joint collagenase paralleled the decline in total nucleated cell count. However, in 3 joints the concentration of collagenase remained high after the total nucleated cell count had returned to normal, suggesting that this enzyme originated from resident articular cells.

Topics: Animals; Arthritis, Infectious; Chromatography, Gel; Collagenases; Enzyme Activation; Horse Diseases; Horses; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases; Neutrophils; Peptide Hydrolases; Phenanthrolines; Phenylmercuric Acetate; Protease Inhibitors; Synovial Fluid; Synovial Membrane

One of the best studied animal models of osteoarthritis is a dog model in which the anterior cruciate ligament of the hind limb stifle joint is transected (Pond-Nuki model). To determine whether stromelysin might play a role in this model, it was necessary to purify the enzyme for production of suitable probes. In the present study, dog synovial fibroblasts were stimulated to express a metalloproteinase that was demonstrated to be canine prostromelysin by Northern blot, protein purification and amino-terminal sequence analyses. Unlike rabbit synoviocytes, passaged dog synoviocytes did not express stromelysin mRNA in response to recombinant human IL-1, but expressed stromelysin mRNA only upon treatment with dog monocyte-conditioned medium (dMCM). The aminophenylmercuric acetate (APMA)-activatable metalloproteinase present in the culture supernatants of stimulated dog synoviocytes was purified using a combination of ion-exchange and dye matrix affinity chromatography. The purified canine metalloproteinase co-migrated on reducing SDS-PAGE with recombinant human prostromelysin-1 as a doublet with apparent molecular masses of 54 and 56 kDa. Similar to APMA-activated human prostromelysin-1, the APMA-activated canine metalloproteinase was inhibited by 1,10-phenanthroline or recombinant human tissue inhibitor of metalloproteinase (TIMP). The amino-terminal sequences of the canine pro- and APMA-activated enzymes were compared with those of human, rabbit and rat stromelysin. The striking homologies among the sequences demonstrated that the purified canine metalloproteinase was indeed canine prostromelysin. A rabbit anti-canine prostromelysin polyclonal antiserum was generated and used to localize the enzyme within cultured dog synoviocytes and articular cartilage stimulated with dMCM. The reagents developed in this study should be useful for examining the expression and distribution of prostromelysin in canine models of osteoarthritis.

Topics: Amino Acid Sequence; Animals; Cartilage, Articular; Cells, Cultured; Collagenases; Culture Media; Cytokines; Disease Models, Animal; Dogs; Enzyme Induction; Enzyme Precursors; Fibroblasts; Glycoproteins; Humans; Leukocytes, Mononuclear; Metalloendopeptidases; Molecular Sequence Data; Osteoarthritis; Phenanthrolines; Phenylmercuric Acetate; Rabbits; Rats; Recombinant Proteins; Sequence Homology; Species Specificity; Stimulation, Chemical; Synovial Membrane; Tissue Inhibitor of Metalloproteinases

Bovine aortic medial tissue and medial smooth muscle cells were demonstrated for the first time to synthesize a latent collagenase together with collagenase inhibitor in culture. Molecular weights of the latent collagenase and its inhibitor derived from aortic medial tissue explant were estimated to be about 52 K by gel filtration and 26.5 K by electrophoresis, respectively. Activated aortic collagenases cleaved type I collagen in solution into 3/4 (alpha A) and 1/4 (alpha B) length cleavage fragments and were inhibited by EDTA, o-phenanthroline, dithiothreitol, bovine serum, and highly purified dental pulp and aortic collagenase inhibitors. The aortic inhibitors showed inhibitory activity against all the animal collagenases tested, except for bacterial collagenase. Double-immunodiffusion analysis using a monospecific antiserum prepared against dental pulp inhibitor showed that the aortic inhibitors are immunologically identical to the pulp inhibitor. Using the same antiserum, we found immunoreactive collagenase inhibitor protein to be localized along the collagen fibers between elastic membranes in aortic medial tissue.

Topics: Animals; Aorta; Blood; Cattle; Cells, Cultured; Chromatography, Gel; Dithiothreitol; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Fluorescent Antibody Technique; Glycoproteins; Immunodiffusion; Immunohistochemistry; Metalloendopeptidases; Microbial Collagenase; Molecular Weight; Muscle, Smooth, Vascular; Phenanthrolines; Phenylmercuric Acetate; Tissue Inhibitor of Metalloproteinases

Human rheumatoid synovial cells in culture stimulated with the conditioned culture medium of rabbit macrophages secrete three distinct latent metalloproteinases. One of them, a proteinase that digests proteoglycan and other connective tissue matrix components, was purified as two active forms after activation with 4-aminophenylmercuric acetate. The two forms were homogeneous on sodium dodecyl sulfate-gel electrophoresis with Mr = 45,000 and Mr = 28,000, whereas the latent precursor was estimated to have Mr = 51,000 by gel permeation chromatography. Both active enzymes had optimal activity at pH 7.5-7.8 and were inhibited by EDTA and 1,10-phenanthroline but not by inhibitors for cysteine, serine, or aspartic proteinases. Removal of Ca2+ from the enzyme solution resulted in a complete loss of activity that could be fully restored by the addition of 1 mM Ca2+. The activity of the apoenzyme was restored by the addition of 0.5 mM Zn2+, 5 mM Co2+, or 5 mM Mn2+ in the presence of Ca2+ but not by each metal ion alone. The identical digestion patterns of reduced, carboxymethylated protein substrates indicated that both active forms of the enzyme have the same substrate specificity. The enzyme degraded cartilage proteoglycans, type I gelatin, type IV collagen, laminin, and fibronectin, and removed the NH2-terminal propeptides from chick type I procollagen. This enzyme may play a role in the normal turnover of the connective tissue matrix as well as in the joint destruction of chronic synovitis.

Topics: Animals; Arthritis, Rheumatoid; Calcium; Cobalt; Connective Tissue; Culture Media; Edetic Acid; Endopeptidases; Extracellular Matrix; Fibroblasts; Humans; Lead; Macrophages; Manganese; Metalloendopeptidases; Molecular Weight; Phenanthrolines; Phenylmercuric Acetate; Protease Inhibitors; Proteoglycans; Rabbits; Synovial Membrane; Zinc

In the transition from proliferation to hypertrophic cell zones in the growth plate, there is an increase in chondrocyte volume and a corresponding decrease in collagen content to accommodate the enlarging cells. It is postulated that collagenase accounts for this collagen loss. To test this hypothesis, tibial growth plates were obtained from normal rats, rachitic rats deficient in vitamin D and phosphate, and rats after 48 and 72 h of healing from rickets. Collagenase was quantitated by a pellet assay based on the release of solubilized collagen from the endogenous insoluble collagen in the tissue homogenates. A fourfold greater collagen release and a concomitant sixfold greater hypertrophic cell volume were measured in rachitic growth plates compared with normal age-matched controls. During healing of rickets, collagenase activity and hypertrophic cell volume returned almost to control levels. Rachitic growth plates were dissected into the juxtaepiphyseal 1/3 and the juxtametaphyseal 2/3. The latter portion contained greater than 95% of the hypertrophic cells and 86% of the collagenase. The collagen-degrading activity was extracted from this region and was shown to be a true collagenase by its production of typical A fragments of tropocollagen produced by collagenase action. The enzyme was activated by aminophenylmercuric acetate and trypsin and was inhibited by EDTA, 1,10-phenanthroline, and a tissue inhibitor of metalloproteinases from human articular cartilage. Inhibitors of aspartic, cysteine, and serine proteases had no effect. Micropuncture fluids aspirated from rachitic cartilage contained latent collagenase activity, indicating an extracellular localization. Negative tests for hemoglobin in the rachitic cartilage samples indicated that there was no contamination by capillaries and that this was not a source of collagenase. It is concluded that extracellular collagenase accounts for the loss of cartilage matrix in the hypertrophic zone, and that this process may be distinct from that of capillary invasion.

Topics: Animals; Cartilage; Electrophoresis, Polyacrylamide Gel; Epiphyses; Male; Microbial Collagenase; Phenanthrolines; Phenylmercuric Acetate; Rats; Rats, Inbred Strains; Rickets; Trypsin; Vitamin D Deficiency

Monolayer and spinner cultured rabbit articular chondrocytes released into the medium latent metal-dependent enzyme with activity against bovine proteoglycan. Pretreatment of medium with p-aminophenylmercuric acetate or trypsin followed by soybean trypsin inhibitor significantly increased enzyme activity. The monolayer-cultured chondrocytes released more of this activity than spinner cultures. The neutral proteoglycanase activity increased with medium concentration and incubation time. Like the human cartilage proteoglycanase, its pH optimum on proteoglycan subunit was 7.25. Gel filtration on BioGel P-30 indicated that the proteoglycanase occurred in two molecular weight forms: 20 000--30 000 and 13 000. The latent enzyme was about 30 000--40 000. The metal-chelators, o-phenanthroline (5 mM) and EDTA (10 mM) inhibited the activated proteoglycanase almost completely, but trypsin and chymotrypsin inhibitors had little effect. The cultured chondrocytes also released into the media a heat-labile inhibitor against the proteoglycanase. The inhibitory activity was present in the nonactivated media and eluted on Sephadex G-100 chiefly at a position corresponding to molecular weights of 10 000--13 000.

Topics: Animals; Cartilage, Articular; Cells, Cultured; Edetic Acid; Endopeptidases; Enzyme Activation; Hydrogen-Ion Concentration; Metalloendopeptidases; Molecular Weight; Phenanthrolines; Phenylmercuric Acetate; Proteoglycans; Rabbits; Trypsin