Purification, kinetic properties and physicochemical characterization of a novel acid phosphatase (AP) from germinating peanut (Arachis hypogaea) seed

Acid phosphatase activity was detected in peanut (Arachis hypogaea) cotyledons during germination. Four (4) to six (6) days of germination was the meantime corresponding to maximum hydrolytic activity of this enzyme. The understanding of the role of acid phosphatase activity during germination led to purify this enzyme by successive chromatography separations on DEAE-Sepharose CL-6B, Sephacryl S-100 HR and Phenyl-Sepharose HP to apparent homogeneity from germinated peanut cotyledon five days old. This enzyme designated peanut cotyledon acid phosphatase (AP) had native molecular weight of 24 kDa by gel permeation. SDS-PAGE of the purified acid phosphatase resolved a single protein band that migrated to approximately 21.5 kDa. Thus, this acid phosphatase likely functions as a monomer. The enzyme had optimum pH (5.0) and temperature (55 degrees C), and appeared to be stable in the presence of anionic, cationic and non-ionic detergents. Substrate specificity indicated that the purified acid phosphatase hydrolyzed a broad range of phosphorylated substrates. However, natural substrates such as ADP and ATP were the compounds with highest rate of hydrolysis for the enzyme. Moreover, the purified acid phosphatase exhibited phytase activity. These results showed that this enzyme played a peculiar role during germination, notably in reducing the rate of phytic acid, an antinutritional substance contained in peanut seed.     

Interrelationship between metabolic and genetic regulation of alkaline and acid phosphatases in E. coli cells]

The effect of exogenous orthophosphate and mutations in regulatory genes of alkaline phosphatase on the level of nonspecific acid phosphatase was studied. The level of this enzyme as well as the level of alkaline phosphatase were shown to be regulated by exogenous orthophosphate being derepressed under phosphate starvation. The derepression of acid phosphatase is accompanied by more rapid secretion of enzyme from membranes to soluble fraction. Mutations in all the four regulatory genes decrease the level of enzyme in cells. Genes phoR and phoS, participating in regulation of alkaline phosphatase, are required for the derepression of acid phosphatase under the conditions of phosphate starvation.     

Isolation and biochemical characteristics of a molecular form of epididymal acid phosphatase of boar seminal plasma

The fluid of boar epididymis is characterized by a high activity of acid phosphatase (AcP), which occurs in three molecular forms. An efficient procedure was developed for the purification of a molecular form of epididymal acid phosphatase from boar seminal plasma. We focused on the epididymal molecular form, which displayed the highest electrophoretic mobility. The purification procedure (dialysis, ion exchange chromatography, affinity chromatography and hydroxyapatite chromatography) used in this study gave more than 7000-fold purification of the enzyme with a yield of 50%. The purified enzyme was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified molecular form of the enzyme is a thermostable 50kDa glycoprotein, with a pI value of 7.1 and was highly resistant to inhibitors of acid phosphatase when p-nitrophenyl phosphate was used as the substrate. Hydrolysis of p-nitrophenyl phosphate by the purified enzyme was maximally active at pH of 4.3; however, high catalytic activity of the enzyme was within the pH range of 3.5-7.0. Kinetic analysis revealed that the purified enzyme exhibited affinity for phosphotyrosine (K(m)=2.1x10(-3)M) and was inhibited, to some extent, by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. The N-terminal amino acid sequence of boar epididymal acid phosphatase is ELRFVTLVFR, which showed 90% homology with the sequence of human, mouse or rat prostatic acid phosphatase. The purification procedure described allows the identification of the specific biochemical properties of a molecular form of epididymal acid phosphatase, which plays an important role in the boar epididymis.     

Purification and characterization of an acid phosphatase that displays phosphotyrosyl-protein phosphatase activity from bovine cortical bone matrix

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 vivo.     

Biosynthesis of acid phosphatase of baker's yeast. Characterization of a protoplast-bound fraction containing precursors of the exo-enzyme.

1. Yeast protoplasts, secreting acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) contain a small amount of firmly bound enzyme, even after lysis (Van Rijn, H.J.M., Boer, P. and Steyn-Parvé, E.P. (1972) Biochim. Biophys. Acta 268, 431-441). The major part (70%) of this protoplast-bound acid phosphatase can be solubilized by nonionic detergents, such as Triton X-100. 2. The kinetics of radioactive amino acid incorporation in the solubilized and in the secreted enzyme has been estimated by pulse-chase labelling of secreting protoplasts, followed by fractionation and counting radioactivity in the enzyme band in polyacrylamide gels after electrophoresis at pH 5.0. A precursor-product relationship between the Triton X-100-extractable fraction of the protoplast-bound acid phosphatase and the secreted enzyme is apparent. 3. The solubilized acid phosphatase is essentially indistinguishable from the secreted enzyme with regard to a number of enzymatic properties and its stability towards pH and temperature. Both enzymes also behave alike on polyacrylamide-gel electrophoresis, producing a single acid phosphatase band with glycoprotein character and comparable mobility. 4. A striking difference is seen in isopycnic equilibrium sedimentation in CsCl: the secreted acid phosphatase is homogeneous, with a buoyant density of p equals 1.47 g/cm3, while the Triton X-100-extractable part of the protoplast-bound acid phosphatase is heterogeneous; besides heavier material a major component with buoyant density of p equals 1.37 g/cm3 is always visible.     

Immunological relatedness of cytoplasmic and membrane-bound acid phosphatases from Dioscorea cayenensis rotundata

Antibodies were raised against one cytoplasmic and two membrane-bound acid phosphatases purified from yam tubers (Dioscorea cayenensis rotundata). Experiments of immunoinactivation and immunoelectrophoresis revealed cross-immunological reactions between the cytoplasmic enzyme (acid phosphatase A) and one of two membrane-bound counterparts (acid phosphatase B) suggesting that these molecules share common antigenic determinants. The antibodies raised against the other membrane-bound enzyme (acid phosphatase C) only inhibited and precipitated this enzyme.     

Analysis of the Acid Phosphatases of Rat Sensory Gangli by Isoelectric Focussing on Polyacrylamide Gels

The acid phosphatases of rat spinal and trigeminal ganglia have been separated by isoelectric focussing on polyacrylamide gels into two main classes with pI's of 7.1 and 5.4. A low-pI acid phosphatase is also present in the mouse but not in the guinea pig. Evidence is presented that in the rat the pI 5.4 enzyme represents the nonlysosomal acid phosphatase that has been identified histochemically in one population of sensory neurones. This pI 5.4 acid phosphatase is partly membrane-bound and is selectively depleted by capsaicin administration. In lumbar dorsal root ganglia the enzyme is selectively depleted by sectioning the sciatic nerve and undergoes rapid axonal transport, a greater amount being transported peripherally than centrally. The results are discussed with reference to the possible function of this nonlysosomal acid phosphatase.     

Ultrastructural localization of acid phosphatase in spermatic cells of Ceratitis capitata (Diptera)

Summary The cytochemical study of acid phosphatase in spermatic cells of Ceratitis capitata defines the enzimatically active sites, relating this enzyme with morphological modifications of the cell components during spermiogenesis. In the axoneme, acid phosphatase is associated with the metabolism of phosphates which promote flagellar motility. The enzymatic activity verified on the cytoplasmic membranes demonstrates the importance of this enzyme in the process of cellular differentiation.     

Oocyte fertilization triggers acid phosphatase activity during Rhodnius prolixus embryogenesis

Acid phosphatase activity, previously identified in Rhodnius prolixus oocytes, was studied during egg development. Fertilized eggs exhibited a five fold increase of total acid phosphatase activity during the first days of development. In contrast non-fertilized oviposited eggs showed no activation of this enzyme. An optimum pH of 4.0 for pNPP hydrolysis in a saturable linear reaction and a strong inhibition by lysosomal acid phosphatase inhibitors such as NaF (10 mM) and Na(+)/K(+) tartrate (0.5 mM) are the major biochemical properties of this enzyme. Fractionation of egg homogenates through gel filtration chromatography revealed a single peak of activity with a molecular mass of 94 kDa. The role of this enzyme in VT dephosphorylation was next evaluated. Western blots probed with anti-phosphoserine polyclonal antibody demonstrated that VT phosphoaminoacid content decreases during egg development. In vivo dephosphorylation during egg development was confirmed by following the removal of (32)P from (32)P-VT in metabolically labeled eggs. Vitellin was the only phosphorylated molecule able to inhibit pNPPase activity of partially purified acid phosphatase. These data indicate that acid phosphatase activation follows oocyte fertilization and this enzyme seems to be involved in VT dephosphorylation.     

Localization of acid phosphatase in Saccharomyces cerevisiae and its export into culture media depends on the type of the N-terminal signal peptide

The aim of this work was to study the character of intracellular distribution and efficiency of yeast acid phosphatase export depending on the type of the N-terminal signal peptide used. A number of plasmids carrying the acid phosphatase genes with different signal peptides sequences was constructed. The main site of the enzyme accumulation for the variant containing its own acid phosphatase signal peptide was the periplasm. Approximately the same pattern was observed when the hybrid signal peptide consisting of acid phosphatase signal peptide and alpha-factor preprosegment tandem was used. Unlike the above-mentioned systems the strain carrying acid phosphatase under the control of alpha factor preprosegment was able to export the enzyme into the culture medium. The experiments have shown the possibility of changing the final localization of secretory proteins by replacing the N-terminal signal peptide.     

Native blot and immunotransfer of human prostatic acid phosphatase isozymes

Agarose gel isoelectrofocusing is used to separate the isozymes of human prostatic acid phosphatase with retention of enzyme activity. The native blotting of the isozymes onto a nitrocellulose membrane increases the sensitivity of the enzyme stain and is suitable for analysis of isozymes in prostate tissue, which contains little nonprostatic acid phosphatase. The specificity of the transfer is increased by treating the membrane with antibody to human prostatic acid phosphatase prior to the transfer. The specificity of the antibody is conferred to the membrane resulting in a transfer specific for prostatic acid phosphatase. The immunotransfer procedure is applicable to serum which contains appreciable amounts of nonprostatic acid phosphatase.     

Modification of the effects of hyperthermia and neutron radiation on the activity of acid phosphatase in CaNT tumors.

Acid phosphatase activity was measured in implanted murine CaNT tumors of varying volumes. There is a clear monotonically increasing relation between acid phosphatase activity and tumor volume. Also the tumors were subjected to either induced artificial hypoxia or hyperthermia (41.0 degrees C) alone, or combined with neutron irradiation (3.8 Gy). Changes in the activity of this enzyme following radiation damage could reflect tissue damage associated with metabolic disturbances. The effect on enzyme activity after sequential hyperthermia and neutron irradiation is not synergistic, as is shown in the quantitative experimental data. This implies that the mechanisms of heat damage differ from that of neutron beam damage, as reflected by acid phosphatase activity. The CaNT tumor was also shown to be thermosensitive after administration of mitoxantrone. Finally, the role of exogenous ATP was shown to provide heat protection by modification of those thermal effects resulting in the activity of acid phosphatase. The augmentation of this hydrolytic enzyme probably represents initial metabolic damage in the tumor after different modalities of radiation alone, or combined with mitoxantrone and exogenous ATP.     

Leishmania donovani: immunochemical localization and secretory mechanism of soluble acid phosphatase

Monoclonal antibodies specific for the soluble, secreted acid phosphatase (EC 3.1.3.2) of Leishmania donovani were used to investigate the localization of this enzyme in extracellular promastigotes and intracellular amastigotes. Indirect immunofluorescence showed a weak general staining in the promastigote cytoplasm, together with strong fluorescence in the flagellar reservoir. Immunofluorescence studies on U937 cells infected in vitro with L. donovani showed that the pathogenic amastigote stage also produced soluble acid phosphatase. Metabolic labeling experiments using promastigotes indicated that the intracellular enzyme was soluble prior to secretion and no evidence was found for the association of secretory acid phosphatase with cell membranes after protein synthesis. The rapid release of acid phosphatase from the flagellar reservoir was energy dependent and may be coupled to beating of the flagellum. The results demonstrated that acid phosphatase was secreted into the flagellar reservoir by Leishmania promastigotes using a conventional constitutive secretory mechanism, and subsequently released from the reservoir into the extracellular medium.     

The development of phosphomonesterases in the testes of prepuberal chicks.

1. The biochemical development and histochemical localisation of phosphomonoesterases in the testes of prepuberal chicks have been studied. 2. Maximum acid phosphatase activity was observed at 12 weeks with a decrease in enzyme activity after this age, whereas alkaline phosphatase activity fluctuated with age. 3. Acid phosphatase activity in chicks was similar to that of the cockerel in being tartarate-insensitive. 4. There was a low level of significant correlation between acid phosphatase activity and testes weight. 5. Both alkaline and acid phosphatase activities were observed in the basement membrane of the seminiferous tubules, and acid phosphatase activity also in the various spermatogenic elements. 6. The results suggest that acid phosphatase is more involved in spermatogenesis, and more widely distributed than alkaline phosphatase in testicular tissue during testicular development.     

Identification and characterization of thiamin repressible acid phosphatase in yeast

We have identified a genetic locus, pho4, in Schizosaccharomyces pombe which encodes a minor expressed cell surface acid phosphatase that is repressed by low concentrations (0.5 microM) of thiamin. The enzyme was purified from a strain that overproduces the enzyme. It is an Asn-linked glycoprotein. Removal of the carbohydrates by endoglycosidase H does not abolish enzymatic activity. The molecular mass of deglycosylated and unglycosylated enzyme that accumulates in membranes when cells are grown in the presence of tunicamycin is 56 kDa as determined by sodium dodecyl sulfate-gel electrophoresis. Thiamin regulation, at least in part, operates by reducing the level of pho4-mRNA. Pho4 is not genetically linked to the phosphate repressible acid phosphatase gene pho1. Phosphate and thiamin repressible acid phosphatase differ in their substrate specificity. Their protein moieties are immunologically related. Pho4 and pho1 are the only genes in S. pombe that express cell surface acid phosphatases being enzymatically active with nitrophenyl phosphate as substrate. S. pombe is not unique in having a thiamin repressible acid phosphatase. In Saccharomyces cerevisiae this enzyme is encoded by PHO3.     

Inhibition of osteoclastic acid phosphatase abolishes bone resorption

Osteoclastic acid phosphatase is a member of a widely-distributed class of iron-containing proteins with acid phosphatase activity. Antibodies raised against one member of this class cross-react with other members from the same or different species, but not with acid phosphatase isoenzymes of different types. When antibodies to one such protein, porcine uteroferrin, are added to medium in which rat osteoclasts are incubated on devitalised cortical bone, both bone resorption and acid phosphatase activity are markedly inhibited. Furthermore, addition of molybdate (an inhibitor of this class of acid phosphatases) also inhibits both bone resorption and enzyme activity. These observations strongly suggest a functional role for osteoclastic acid phosphatase in bone resorption.     

Processing, transport, and secretion of the lysosomal enzyme acid phosphatase in Dictyostelium discoideum

To explain the different secretion kinetics of lysosomal enzymes in Dictyostelium discoideum, previous investigators have hypothesized the existence of a heterogeneous population of lysosomes containing either the enzyme acid phosphatase or other hydrolase enzymes. This proposal predicts that at least two targeting mechanisms exist for lysosomal enzymes in this organism. To begin to investigate this possibility, the transport, processing, and targeting of acid phosphatase was studied by using a combination of radiolabel pulse-chase procedures, subcellular fractionations, and indirect immunofluorescence microscopy. Acid phosphatase was initially synthesized in axenically growing cells as a 56-kDa precursor polypeptide that was proteolytically processed after 20 min to a 55-kDa mature protein. This enzyme was rapidly transported from the endoplasmic reticulum to Golgi complex (halftime of 3 min) as measured by the acquisition of resistance to the enzyme endoglycosidase H. Furthermore, Percoll gradient fractionations indicated that radiolabeled forms of acid phosphatase reached dense lysosomal vesicles at about the same time as final processing was occurring. Proper sorting of acid phosphatase in D. discoideum apparently was not critically dependent on low intravacuolar pH since the addition of ammonium chloride did not stimulate the missorting and secretion of acid phosphatase. These results are very similar to previous observations concerning other Dictyostelium lysosomal enzymes. Consistent with the existence of a heterogeneus population of lysosomes, the percentage of radiolabeled acid phosphatase secreted 4 h into a chase period was 15-fold lower as compared with another lysosomal enzyme, beta-glucosidase. However, acid phosphatase, alpha-mannosidase, and beta-glucosidase were all predominantly colocalized as determined by indirect immunofluorescence, which for the first time demonstrates the homogeneous nature of the lysosomal system in D. discoideum. Taken together these results suggest that the processing and transport of acid phosphatase may be similar in nature to the glycosidases. However, the different kinetics of secretion of acid phosphatase versus the colocalized glycosidase enzymes suggests that an undefined mechanism operates to distinguish these classes of enzymes at a step after localization to lysosomes but prior to secretion.     

Biochemical and genetic evidence that yeast extracellular protein phosphatase activity is due to acid phosphatase

In this paper evidences are presented strongly confirming that an extracellular 32P-phosphopeptide phosphatase activity of yeast is accounted for by acid phosphatase. Dephosphorylation of 32P phosphoseryl peptides was achieved with whole yeast cells, thus demonstrating extracellular location of protein phosphatase activity. The acid phosphatase and protein phosphatase activity copurified throughout purification procedure. Purified enzyme showed the same pH-profile and had the same Km value with phosphopeptide substrate as intact cells. Protein phosphatase activity is repressed by phosphate in the same manner as acid phosphatase activity, showing that not only repressible but also constitutive acid phosphatase displays protein phosphatase activity. Using mutant strains defective in acid phosphatase activity it was confirmed that acid phosphatase and protein phosphatase activities are the products of the same gene(s).     

Formation of secreted acid phosphatase during the growth of Saccharomyces cerevisiae yeasts on different sources of carbon and nitrogen nutrition

The effect of certain components in the growth medium on the secretion of acid phosphatase was studied with Saccharomyces cerevisiae. The presence of phosphate at a concentration of 10 mM in the medium inhibited the formation of repressible forms of this enzyme. The synthesis of the secreted enzyme depended on the sources of carbon and nitrogen nutrition. The enzyme yield was highest in a medium with sucrose as a carbon source and ammonium chloride as a nitrogen source. The secretion of acid phosphatase is stimulated by an increase in the sugar content and a deficiency of the nitrogen source in the medium.     

Isolation and biochemical characteristics of a molecular form of epididymal acid phosphatase of boar seminal plasma

The fluid of boar epididymis is characterized by a high activity of acid phosphatase (AcP), which occurs in three molecular forms. An efficient procedure was developed for the purification of a molecular form of epididymal acid phosphatase from boar seminal plasma. We focused on the epididymal molecular form, which displayed the highest electrophoretic mobility. The purification procedure (dialysis, ion exchange chromatography, affinity chromatography and hydroxyapatite chromatography) used in this study gave more than 7000-fold purification of the enzyme with a yield of 50%. The purified enzyme was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified molecular form of the enzyme is a thermostable 50 kDa glycoprotein, with a pI value of 7.1 and was highly resistant to inhibitors of acid phosphatase when p-nitrophenyl phosphate was used as the substrate. Hydrolysis of p-nitrophenyl phosphate by the purified enzyme was maximally active at pH of 4.3; however, high catalytic activity of the enzyme was within the pH range of 3.5–7.0. Kinetic analysis revealed that the purified enzyme exhibited affinity for phosphotyrosine (K_m = 2.1 × 10⁻³ M) and was inhibited, to some extent, by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor. The N-terminal amino acid sequence of boar epididymal acid phosphatase is ELRFVTLVFR, which showed 90% homology with the sequence of human, mouse or rat prostatic acid phosphatase.The purification procedure described allows the identification of the specific biochemical properties of a molecular form of epididymal acid phosphatase, which plays an important role in the boar epididymis.