acid-phosphatase and beta-glycerophosphoric-acid

Cryptococcus neoformans strains isolated from patients with AIDS secrete acid phosphatase, but the identity and role of the enzyme(s) responsible have not been elucidated. By combining a one-dimensional electrophoresis step with mass spectrometry, a canonically secreted acid phosphatase, CNAG_02944 (Aph1), was identified in the secretome of the highly virulent serotype A strain H99. We created an APH1 deletion mutant (Δaph1) and showed that Δaph1-infected Galleria mellonella and mice survived longer than those infected with the wild type (WT), demonstrating that Aph1 contributes to cryptococcal virulence. Phosphate starvation induced APH1 expression and secretion of catalytically active acid phosphatase in the WT, but not in the Δaph1 mutant, indicating that Aph1 is the major extracellular acid phosphatase in C. neoformans and that it is phosphate repressible. DsRed-tagged Aph1 was transported to the fungal cell periphery and vacuoles via endosome-like structures and was enriched in bud necks. A similar pattern of Aph1 localization was observed in cryptococci cocultured with THP-1 monocytes, suggesting that Aph1 is produced during host infection. In contrast to Aph1, but consistent with our previous biochemical data, green fluorescent protein (GFP)-tagged phospholipase B1 (Plb1) was predominantly localized at the cell periphery, with no evidence of endosome-mediated export. Despite use of different intracellular transport routes by Plb1 and Aph1, secretion of both proteins was compromised in a Δsec14-1 mutant. Secretions from the WT, but not from Δaph1, hydrolyzed a range of physiological substrates, including phosphotyrosine, glucose-1-phosphate, β-glycerol phosphate, AMP, and mannose-6-phosphate, suggesting that the role of Aph1 is to recycle phosphate from macromolecules in cryptococcal vacuoles and to scavenge phosphate from the extracellular environment.. Infections with the AIDS-related fungal pathogen Cryptococcus neoformans cause more than 600,000 deaths per year worldwide. Strains of Cryptococcus neoformans isolated from patients with AIDS secrete acid phosphatase; however, the identity and role of the enzyme(s) are unknown. We have analyzed the secretome of the highly virulent serotype A strain H99 and identified Aph1, a canonically secreted acid phosphatase. By creating an APH1 deletion mutant and an Aph1-DsRed-expressing strain, we demonstrate that Aph1 is the major extracellular and vacuolar acid phosphatase in C. neoformans and that it is phosphate repressible. Furthermore, we show that Aph1 is produced in cryptococci during coculture with THP-1 monocytes and contributes to fungal virulence in Galleria mellonella and mouse models of cryptococcosis. Our findings suggest that Aph1 is secreted to the environment to scavenge phosphate from a wide range of physiological substrates and is targeted to vacuoles to recycle phosphate from the expendable macromolecules.

Topics: Acid Phosphatase; Acquired Immunodeficiency Syndrome; Animals; Biological Transport; Cryptococcosis; Cryptococcus neoformans; Disease Models, Animal; Female; Fungal Proteins; Gene Deletion; Glucosephosphates; Glycerophosphates; Green Fluorescent Proteins; Humans; Hydrogen-Ion Concentration; Mannosephosphates; Mice; Mice, Inbred BALB C; Monocytes; Moths; Proteomics; Vacuoles

The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using β-glycerophosphate (β-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of β-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of β-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca(2+) present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. β-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (Pi). Levamisole at the IC50 spared significantly parasite growth when β-GP was the sole source of Pi and stopped it in the absence of β-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle.

Topics: Acid Phosphatase; Alkaline Phosphatase; Catalysis; Cations, Divalent; Glycerophosphates; Hydrogen-Ion Concentration; Hydrolysis; Levamisole; Nitrophenols; Organophosphorus Compounds; Substrate Specificity; Trypanosoma rangeli

A study of the histochemical reaction for acid phosphatase (AcPase) in venom gland secretory cells from Bothrops jararaca was done to investigate the distribution of lysosomes and related structures in stages of high- and low-protein synthesis. From this analysis, it was expected to gain insight into the cellular pathway by which AcPase is secreted into the venom. Two subtypes of AcPase reactivities were detected in the venom gland secretory cells: one was found in lysosomes and related structures and in some trans-Golgi network (TGN) elements and reacts with beta-glycerophosphate (betaGP) as substrate; the other was found in secretory vesicles, apical plasmalemma, lysosomes and related structures, and in some TGN elements, and reacts with cytidine monophosphate (CMP). The results are compatible with the possibility that there is a secretory via for AcPase in the venom gland of B. jararaca and that the elements composing this pathway are noted only when CMP is used as substrate. Large autophagosomes reactive to both betaGP and to CMP were commonly observed in the basal region of the secretory cells, and they were more abundant in the glands during the stage of low activity of protein synthesis.

Topics: Acid Phosphatase; Animals; Bothrops; Cell Membrane; Cytidine Monophosphate; Glycerophosphates; Golgi Apparatus; Histocytochemistry; Lysosomes; Secretory Vesicles; Substrate Specificity; Tissue Distribution; Venoms

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

The acid phosphate activity (APA) associated with the isolated brush border membrane of the tapeworm, Hymenolepis diminuta, hydrolyzed p-nitrophenyl phosphate (PNPP), pyrophosphate (PPi), and beta-glycerophosphate (beta GP). Inhibition of PNPP hydrolysis at pH 4.0 was inhibited in a competitive manner by the following compounds (listed in order of decreasing affinity with their apparent inhibitor constants (Ki')): molybdate (0.031 mM); PPi (0.147 mM); NaF (0.150 mM); o-carboxyphenyl phosphate (0.261 mM); inorganic phosphate (0.770)); arsenate (3.45 mM); tartrate (22.1 mM); and beta GP (29.8 mM). Cu2+, formaldehyde, and arsenite at 10:1, 80:1, and 200:1 inhibitor to substrate ratios did not inhibit APA. The maximal rate of hydrolysis (Vmax) of each substrate was greater at pH 4.0 than 5.0. The apparent Michaelis constant (Km') for PNPP increased from 0.233 to 0.351 mM when the pH was raised from 4.0 to 5.0. The Km' for PPi decreased from 0.101 to 0.046 mM, while the Km' for beta GP changed from 2.04 to 2.22 mM under similar circumstances. APA and alkaline phosphatase activity increased as a function of temperature up to 45 degrees C.

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Cell Membrane; Diphosphates; Glycerophosphates; Hydrogen-Ion Concentration; Hymenolepis; Kinetics; Microvilli; Nitrophenols; Organophosphorus Compounds; Temperature

An acid phosphatase activity has been detected in purified preparations of African swine fever virus. Purified viral cores obtained after Nonidet-P40 and 2-mercaptoethanol treatment of the virus retained the activity as assayed with nitrophenyl phosphate as substrate at pH 5. Enzyme cytochemistry by electron microscopy showed that the acid phosphatase activity is localized mainly inside the core of the virion. The molecular weight and the isoelectric point of the virus acid phosphatase activity confirmed that it was distinct from the host cellular enzyme.

Topics: Acid Phosphatase; African Swine Fever Virus; Glycerophosphates; Iridoviridae; Isoelectric Point; Molecular Weight; Virion

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

A cytochemical study of acid phosphatase (AcP-ase) in the lateral prostate of the rat was performed to investigate whether AcP-ase in the secretory apparatus can be distinguished from AcP-ase in lysosomes and their related structures. Two types of AcP-ase were observed in the rat lateral prostate. One was found in the secretory apparatus (Golgi saccules and some Golgi vesicles, condensing and secretory vacuoles), and reacted well with naphthol AS-BI phosphate (N AS-BI P) as substrate; the other was found in the lysosomes and Golgi-associated endoplasmic-reticulum-lysosome system (GERL)-like structure, and reacted well with beta-glycerophosphate (beta GP) as substrate. Although the AcP-ase which reacted well with N AS-BI P was also observed in certain portions of pleomorphic lysosomes, it was concluded that it was the same as the AcP-ase found in the condensing and secretory vacuoles, since a lysosome engulfing a condensing vacuole was often observed. Therefore, it is concluded that the AcP-ase in the secretory apparatus in the rat lateral prostate is different from the AcP-ase in lysosomes. Condensing vacuoles appear to originate from particular portions of Golgi saccules, but not from the GERL or GERL-like structure, at least in the rat lateral prostate.

Topics: Acid Phosphatase; Animals; Cytoplasmic Granules; Endoplasmic Reticulum; Glycerophosphates; Golgi Apparatus; Histocytochemistry; Lysosomes; Male; Organophosphorus Compounds; Prostate; Rats; Rats, Inbred Strains; Vacuoles

The subcellular localization of acid phosphatase (AcP) in various immunologically-defined neoplastic lymphoid cells including hairy cells was investigated by electron microscopy. 2 substrates, naphthol-AS-BI-phosphoric acid (naphthol-AS-BI-P) and sodium beta-glycerophosphate, were compared. By incubation in naphthol-AS-BI-P containing medium, the reaction product was found located in granules, vesicles, the Golgi apparatus, the rough ER including the nuclear envelope in the cells of T ALL, T CLL and HCL. A typical pattern of reaction was observed for each of these disorders: enzyme-positive Golgi membranes and neighbouring granules, clustered in the nuclear notch in T cell-derived lymphoblasts; enzyme-positive granules around Gall bodies, aggregated paranuclearly in T CLL lymphocytes and enzyme-positive scattered cytoplasmic granules and vesicles in hairy cells. Enzyme activity was occasionally seen in singly-occurring granules in the cells of cALL, B CLL and B PLL, rarely in other substructures. With the Gomori method using beta-glycerophosphate as substrate, the enzyme reaction was limited primarily to lysosomal sites and was seldom observed in other organelles. Tartrate-resistant AcP was found in the majority of hairy cells and in a few prolymphocytes, located in the same structures as AcP without tartrate.

Topics: Acid Phosphatase; B-Lymphocytes; Cytoplasmic Granules; Glycerophosphates; Golgi Apparatus; Humans; Leukemia, Hairy Cell; Leukemia, Lymphoid; Microscopy, Electron; Organophosphorus Compounds; T-Lymphocytes

In this study acid phosphatase (ACPase) was localized in the retinal pigment epithelium (RPE) of normal and Royal College of Surgeons (RCS) rats pink-eyed and pigmented with inherited retinal dystrophy to determine differences in staining during the post-engulfment stages of phagocytosis using two substrates, Na-beta-glycerophosphate and cytidine-5'-monophosphate. Staining was similar using either substrate and in the normal RPE the Golgi system, lysosomes and phagosomes were ACPase-positive. In the dystrophic RPE, which has a diminished capacity to phagocytose photoreceptor rod outer segments, ACPase staining was localized on melanosomes in the pigmented dystrophic and on the apical microvillous membranes in the pink-eyed dystrophic, but was not localized on phagosomes in either the pink-eyed or pigmented dystrophic RPE. Since only a few phagosomes were seen at any given time in dystrophic RPE in vivo, a tissue explant system was used to examine the number of latex beads phagocytosed by normal and RCS RPE, as well as the number of phagosomes containing both beads and ACPase activity in the normal and mutant RPE. Our findings indicate that in the dystrophic, fewer phagosomes are ACPase-positive than in the normal, and that some enzyme may be inappropriately shunted to either the apical microvilli or to melanosomes instead of to phagolysosomes.

Topics: Acid Phosphatase; Animals; Cytidine Monophosphate; Glycerophosphates; Histocytochemistry; Microscopy, Electron; Organoids; Phagocytes; Pigment Epithelium of Eye; Rats; Retinal Degeneration; Substrate Specificity

Topics: Acid Phosphatase; Animals; Bone and Bones; Enzyme Activation; Femur; Glycerophosphates; Nitrophenols; Organophosphorus Compounds; Potassium Chloride; Rats; Sucrose; Tibia

Topics: Acid Phosphatase; Animals; Axons; Brachial Plexus; Cats; Cervical Cord; Cytoplasm; Diphosphates; Glycerophosphates; Golgi Apparatus; Histocytochemistry; Hydrolysis; Lysosomes; Mitochondria; N-Glycosyl Hydrolases; Neurons; Neurosurgery; Nucleosides; Phosphoric Monoester Hydrolases; Research; Spinal Cord; Thiamine Pyrophosphate