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PubMed1981

Acid phosphatase isoenzymes of Chlamydomonas reinhardii.

Acid phosphatase isoenzymes of Chlamydomonas reinhardii were investigated by isoelectric focusing in polyacrylamide gel systems. In this paper we describe in detail an original method for isoelectric focusing of acid phosphatases extracted from wild-type and acid phosphatase-lacking mutant algae, obtained from Laboratoire de Génetique of University of Liège. Three isoenzymes can be separated from the buffer-soluble components of these cells. An additional isoenzyme type can be visualized using the nonionic detergent NP40 as solubilizer. We conclude that these four isoenzymes are related to the structural gene of the soluble constitutive acid phosphatase, which was shown by their appearance in P2 and their total absence in mutant Pa. The pI values of soluble constitutive acid phosphatase isoenzymes range between pH 5.2 and 6.2. As a result of treatment with NP40 the extracts from both wild-type and mutant lines contain two additional active phosphatase forms which can be characterized by their high heat resistance and low pI values. These enzymes are fully active using either alpha-naphthyl phosphate or different acetate esters as substrates.

Acid Phosphatase↗
PubMed1975

Enhancing effect of surfactant and protein on hydrolysis of thymolphthalein monophosphate by purified prostatic acid phosphatase.

Purified prostatic acid phosphatase catalyzes the hydrolysis of thymolphthalein monophosphate 10-fold faster if an optimal concentration of Brij 35 (a wetting agent) or protein (bovine serum albumin or human serum proteins) is present. Results of gel filtration, dialysis, and sucrose density-gradient centrifugation analysis suggest that the substrate must combine with detergent or protein before the enzyme can catalyze its hydrolysis.

Acid Phosphatase↗
PubMed1975

The insignificance of an elevated acid phosphatase in children.

Acid phosphatase values in normal children were incorrectly assumed to be within the same range of adult levels. The enzyme was found to be elevated in 25 boys less than 14 years old and did not return to adult levels until after puberty. It is emphasized that extensive genitourinary investigation and prostatic biopsy are not indicated on the basis of elevated enzyme levels in the prepuberal age group.

Acid Phosphatase↗
PubMed1976

Effect of salts on the kinetic parameters and thermal stability of bovine brain acid phosphatase.

Bovine brain acid phosphatase is inhibited, at any pH, by an increase in ionic strength. The rate decrease is associated at pH 5, with a marked decrease in Km and, at pH 8, with a noticeable decrease in Vm. The rate of thermal inactivation of the enzyme is unaffected by increasing ionic strength up to 300 mM. These results are discussed in terms of interactions at the active site of the enzyme.

Acid Phosphatase↗
PubMed1991

Homodimer and heterodimer subunits of human prostate acid phosphatase.

Human prostatic acid phosphatase (PAP) isoenzymes, designated PAP-A and PAP-B, were isolated from human seminal plasma by sequential affinity chromatography on concanavalin A and L(+)-tartrate, a classic inhibitor of PAP. Both the major PAP-A and the minor PAP-B isoenzymes exhibited a similar molecular mass (100 and 105 kDa respectively), multiple pI values (5.05-5.35 and 5.05-5.12), and substrate and inhibitor specificity. Immunological characterization revealed that PAP-B possesses distinct antigenic determinants, in addition to the common sites shared with PAP-A. SDS/PAGE indicated that both isoenzymes are composed of two subunits of 50 kDa each. At high salt concentration, PAP-B dissociated completely into single subunits of 50 kDa, whereas PAP-A remained intact at 100 kDa. PAP-B was resolved by reverse-phase h.p.l.c. into three components, designated alpha, beta and gamma, each of 50 kDa, at a molar ratio of approx. 2:1:1. PAP-A contained a single component of molecular mass 50 kDa. The single component of PAP-A and the alpha component of PAP-B possessed identical amino acid compositions and N-terminal sequences, which were different from those of the beta and gamma components. These results indicate that human PAP contains three isoforms, alpha 2, alpha beta and alpha gamma. PAP-A, the major isoenzyme, is a homodimer consisting of two identical subunits (alpha 2), and PAP-B, the minor isoenzyme, is a mixture of two heterodimers, consisting of non-identical subunits (alpha beta and alpha gamma).

Acid Phosphatase↗
PubMed1978

Purification and characterization of human prostatic acid phosphatase.

Human prostatic acid phosphatase (orthophosphoric monoester phosphohydrase, EC 3.1.3.2) is purified to homogeneity by standard procedures which include CM-Sephadex, Con A affinity chromatography and gel filtration. The purified enzyme is antigenically specific and has a M.W. of 100,000 with subunit M.W. of 48,000. However, the enzyme exhibited charge heterogeneity. Two major electrophoretic or chromatographic isozymic forms of PAP were separated by DEAE-Sephadex chromatography and their immunochemical identity was studied by immunodiffusion before and after the neuraminidase digestion. Quantitative precipitin and inhibition experiments showed immunological identity of the two chromatographic isozymes. Immunologic specificity of this enzyme resides on the protein moiety rather than the carbohydrate residue, although the latter group is mostly responsible for the charge group heterogeneity of the enzyme.

Acid Phosphatase↗
PubMed2000

Crystal structure of human prostatic acid phosphatase .

BACKGROUND: Prostatic acid phosphatase (hPAP) is a major product of the human prostate gland, yet its physiological substrate remains unknown. METHODS: Human PAP, purified from semen, was crystallized using polyethylene glycol as the precipitant and its crystal structure was determined using X-ray diffraction. The structure was refined at 3.1 A resolution to R = 16% and R(free) = 27%. RESULTS: The structure of hPAP is similar to that of other known histidine phosphatases, and the positions of its catalytic residues are conserved. N-linked carbohydrates are present at each of the possible glycosylation sites. It appears that high-mannose chains are attached to Asn 62 and Asp 301, while complex chains are at Asn 188. CONCLUSIONS: The similarity of the three-dimensional structures of rat PAP and human PAP indicates that the mechanistic analyses of the catalytic mechanism proposed for the rat enzyme should be extended to the human enzyme without reservations. The crystallographic data allowed the correlation of attachment sites of N-linked carbohydrate chains with a given carbohydrate type. The carbohydrates of the protein produced in the prostate cells and in the baculovirus expression system appear to differ at the site of complex carbohydrates attachment.

Acid Phosphatase↗
PubMed1996

The folding intermediate of reversibly denatured human prostatic acid phosphatase.

Human prostatic acid phosphatase (hPAP) [EC 3.1.3.2], a homodimer of ca. 50 kDa subunit molecular weight, shows reversible denaturation in 6 M urea at pH 2.5. Rapid dilution of the denatured enzyme allowed partial renaturation of hPAP, as measured by enzyme activity, to a level which depended on the composition of the dilution solution employed and time of the reaction. The renaturation reaction of hPAP was examined using spectral analysis (circular dichroism and fluorometry), fast size-exclusion chromatography and proteolysis by trypsin. The observed results are in agreement with the concentration-dependent kinetics of hPAP reactivation, assuming that the reconstitution of the active enzyme requires the association of subunits in dimeric form. Moreover, it suggests formation of an inactive intermediate during refolding of the denatured PAP. A mechanism of renaturation of the active enzyme from denatured PAP is proposed.

Acid Phosphatase↗
PubMed1996

Tyrosine phosphorylation of a 185 kDa phosphoprotein (pp185) inversely correlates with the cellular activity of human prostatic acid phosphatase.

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.

Acid Phosphatase↗
PubMed1990

Intrauterine growth: association with acid phosphatase genetic polymorphism.

Acid phosphatase (ACP1) is an enzyme found in the cytoplasm of many tissues and probably functions as a flavin mononucleotide-phosphatase. Therefore the highest concentration of flavin-mononucleotide cofactors is expected in ACP1 phenotypes with the lowest enzymatic activity (A and BA) and the lowest concentration of these cofactors is expected in phenotypes with the highest activity (CB and C). Accordingly, metabolic activities related to flavoenzymes should attain maximal levels in A and BA phenotypes and minimal levels in CB and C phenotypes. In the present study we have analyzed possible effects of ACP1 genetic variability on intrauterine growth in a sample of 609 newborns collected from three consecutive series in Rome. An association between ACP1 and birth weight is observed. The association is present only among male infants. ACP1 phenotypes with low enzymatic activity (A and BA) show a clear tendency to higher rates of intrauterine growth. A linear negative correlation is also observed between enzymatic activity and quartile class. The relation is significant only in male infants. The data suggest that in fetuses with low ACP1 activity, metabolic activity may be regulated at a level allowing a full response to specific genetic stimuli maximizing fetal growth.

Acid Phosphatase↗
PubMed1993

Fluorometric analysis of native, urea-denatured and refolded human prostatic acid phosphatase.

Human prostatic acid phosphatase (EC 3.1.3.2) was denatured in 6 M urea at pH 2.5, but was refolded by dilution at pH 7.0, as demonstrated by the recovery of nearly complete enzyme activity and dimeric structure. The conformational changes among the native, denatured and refolded states were monitored by means of steady-state and nanosecond pulse fluorometry of tryptophan residues of the enzyme. The relative quantum yield of the fluorescence was highest in the native enzyme and lowest in the denatured one, and was intermediate in the refolded enzyme, although the emission peak was reproducible after refolding. The observed decay curves of tryptophan fluorescence of the native, denatured and refolded states were analyzed by decay functions of three lifetimes. The fluorescence lifetimes of the refolded enzyme were shorter than those of the native one. The fluorescence of the denatured enzyme decayed much faster than that of the other forms. The fluorescence excitation spectra revealed that the excitation energy of phenylalanine was transferred to tryptophan(s) in the native and refolded forms, but not in the denatured form. The efficiency of the energy transfer was higher in the native enzyme than in the refolded one. It was found by excitation polarization spectra that the freedom of internal motion of tryptophans was greater in the refolded enzyme than in the native enzyme. In the denatured enzyme the polarization anisotropies were very low. These results indicate that the higher structure with respect to tryptophans of the refolded enzyme is delicately but definitely different from that of the native enzyme and that local conformation of the active center is recovered upon refolding.

Acid Phosphatase↗
PubMed2001

Mutational analysis of the interaction between active site residues and the loop region in mammalian purple acid phosphatases.

Mammalian purple acid phosphatases (PAPs) can be divided into two groups, which exhibit distinct spectroscopic and kinetics properties: PAPs that consist of a single 36 kDa polypeptide, and PAPs that have undergone limited proteolysis to give two fragments with masses of 16 and 20 kDa, respectively. Proteolysis results in an increase in enzymatic activity, an increase in the optimal pH for activity, and a change in the g(z)() value of the characteristic EPR spectrum of the mixed-valence binuclear iron center. It has been proposed that these changes are due to the loss of interactions between Asp146 in an exposed loop region and active site residues upon proteolysis. In the present study, site-directed mutagenesis of Asp146 in recombinant rat bone PAP (recRPAP) has confirmed this hypothesis. Conversion of Asp146 into Ala, which eliminates the interaction of the side chain with the active site, resulted in an enzyme with properties typical of PAPs isolated in proteolytically cleaved forms. The Asp146Asn and Asp146Glu mutants were also prepared and examined to assess the effects of altered electrostatic interactions and side-chain length. Limited proteolysis of all three mutant enzymes with cathepsin L resulted in a significant increase in catalytic activity. Thus, although the interaction between Asp146 and (an) active site residue(s) is the major factor responsible for the low catalytic activity of uncleaved PAPs, other interactions are also important. Since both p-nitrophenyl phosphate and osteopontin, a potential in vivo substrate, show the same level of activation, the observed increase in catalytic activity upon proteolysis is likely to be due to electrostatic rather than steric effects. EPR spectra of FeZn-recRPAP before and after cleavage by cathepsin L suggest that cleavage primarily affects the divalent metal site. The observation that pK(es,1) is also sensitive to changes at the divalent site is consistent with the proposal that the nucleophilic hydroxide is that bridging the divalent and trivalent metals.

Acid Phosphatase↗
PubMed2005

Safety and immunological efficacy of a prostate cancer plasmid DNA vaccine encoding prostatic acid phosphatase (PAP).

Prostatic acid phosphatase (PAP) is a prostate tumor antigen currently being investigated as a target antigen in several human vaccine trials, some with evidence of clinical benefit. We have previously demonstrated that plasmid DNA vaccines encoding either human or rat PAP can elicit antigen-specific cellular and humoral immunity in rat models. The current study was performed to determine the safety and potential immunological efficacy in rodents of large and repetitive doses of a GMP-grade plasmid DNA vaccine encoding human PAP, pTVG-HP. Fifty-four male Lewis rats were immunized intradermally at 2-week intervals with 100, 500, or 1,500 microg pTVG-HP with 5 microg recombinant rat GM-CSF protein given as a vaccine adjuvant. An additional 12 male Lewis rats served as controls with groups immunized with 1,500 microg of a parental DNA vector not encoding human PAP, and a group that received GM-CSF protein only without plasmid DNA. Groups of animals (n=3-6) were euthanized after two, four, or six immunizations with collections of tissues and blood for toxicity assessment and immunological analysis. No significant toxicities were observed in terms of animal weights, histopathology, hematological changes, or changes in serum chemistries. Six of fifty-four were found to have subtle evidence of possible renal toxicity, however these findings were not statistically different from control animals. The vaccine was found to be effective in eliciting PAP-specific CD4 and CD8 T cells, predominantly Th1 in type, in all immunized animals at all doses and numbers of immunizations. PAP-specific IgG were detected in a dose-dependent fashion, with titers increasing after multiple immunizations. These studies demonstrate that, in rats, immunization with the pTVG-HP vaccine is safe and effective in eliciting PAP-specific cellular and humoral immune responses. These findings support the further clinical evaluation of pTVG-HP in patients with prostate cancer.

Acid Phosphatase↗
PubMed2002

Characterization of a phosphatase secreted by Staphylococcus aureus strain 154, a new member of the bacterial class C family of nonspecific acid phosphatases.

An acid phosphatase, designated SapS, hydrolyzing p-nitrophenyl phosphate (pNPP), was identified and characterized from the culture supernatant of a Staphylococcus aureus strain isolated from vegetables. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the protein indicated an estimated molecular mass of 30 kDa. The enzyme displayed optimum activity at 40 degrees C and pH 5. Characterization of the phosphatase in a reconstitution assay showed that MgCl2 and Triton X-100, respectively, restored maximal activity, but not CaCl2 The phosphatase activity was affected by EDTA and sodium molybdate. The DNA sequence encoding SapS was cloned and sequenced. The putative acid phosphatase gene encodes a protein of 296 amino acids with a 31-residue signal peptide. Database searches revealed significant structural homology of SapS to several proteins belonging to the bacterial class C family of nonspecific acid phosphatases. Comparison of the sequences indicated that despite a low level of overall conservation between the proteins, four conserved sequence motifs could be identified.

Acid Phosphatase↗
PubMed1985

Characterization of Fe2+-activated acid phosphatase in rat epidermis.

A particulate acid phosphatase (EC 3.1.3.2, orthophosphoric monoester phosphohydrolase (acid optimum)) was extracted in 1 M KCl, from 2-day rat epidermis. The enzyme has a Mr of 32,000, but two forms, F1 and F2 with pI values of 8.6 and 8.3, respectively, were identified while the pI values of other acid phosphatases soluble in sucrose and Triton X-100 were all acidic. F1 and F2 also differed from other epidermal acid phosphatases because they were (a) activated by Fe2+ and reducing agents, (b) showed immunological cross-reactivity with purple acid phosphatase of rat spleen and (c) dephosphorylated phosvitin and alpha-casein even though they had rather high Km values.

Acid Phosphatase↗
PubMed1978

Isoenzymes of human prostate acid phosphatase.

The isoenzymes of human prostatic acid phosphatase have been studied by an isoelectric focusing technique. Purified acid phosphatase from malignant prostates contained eight isoenzymes with pI 4.4--5.3. The sera from patients with prostate cancer were shown to have similar acid phosphatase isoenzyme patterns at pI 4.0--5.5; as the serum enzyme activities increased, the pI of isoenzymes shifted to more acidic pH. These isoenzyme patterns of sera from patients with prostate cancer were different from those of patients with Gaucher's disease or from acid phosphatase of human erythrocytes, both of which exhibited only one enzyme band around pI 5.0 and 6.0, respectively. Treatment of serum sample of prostate cancer with neuraminidase did not result in a single enzyme band but alter the pI of isoenzymes, which shifted to a higher pH region. The significance of acid phosphatase activities and its isoenzyme patterns in prostate cancer merits further investigation.

Acid Phosphatase↗
PubMed1992

The pho-2A mutant of Neurospora crassa which is deficient in Pi-repressible alkaline phosphatase (EC 3.1.3.1) is also defective in Pi-repressible acid phosphatase (EC 3.1.3.2).

1. The mycelial Pi-repressible acid phosphatase presented p-nitrophenylphosphatase activity with negative cooperativity and Michaelian behavior when synthesized by the wild-type and pho-2A mutant strains of Neurospora crassa, respectively. 2. The major acid phosphatase present in cell extracts of the pho-2A mutant of N. crassa grown in low Pi medium is more thermolabile (t1/2 = 4 min at 54 degrees C, pH 5.4) than that of the wild strain (stable for at least 80 min at 54 degrees C, pH 5.4). 3. The pho-2A mutant of N. crassa secreted a more thermolabile acid phosphatase (t1/2 = 30 min at 50 degrees C, pH 5.4) than the wild strain (t1/2 of at least 80 min at 50 degrees C, pH 5.4). 4. The pho-2A mutant of N. crassa synthesized a more thermolabile acid phosphatase (t1/2 = 37 min at 54 degrees C, pH 5.4) than the wild strain in high Pi medium (t1/2 = 14 min at 54 degrees C, pH 5.4). 5. The pleiotropic nature of the pho-2 locus and its possible involvement in the mechanism of phosphatase secretion by N. crassa are proposed.

4-Nitrophenylphosphatase↗