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Because tryptophans are found as part of the phosphate binding sites in a number of proteins, human prostatic acid phosphatase (hPAP) was examined for the presence and the role of essential tryptophan residues. The pH dependence of the intrinsic fluorescence of hPAP resembled the kinetic pH dependence. Chemical modification by N-bromosuccinimide (NBS) resulted in an inactivation of the enzyme and produced a characteristic reduction of the protein absorbance at 280 nm. Two tryptophans per subunit were modified, and this was accompanied by an apparently complete loss of enzymatic activity. In the presence of the competitive inhibitor L-(+)-tartrate, the loss of enzyme activity was significantly reduced as compared to the rate of tryptophan modification. After labeling the protein with 2,4-dinitrophenylsulfenyl chloride (DNPS-Cl), two tryptic peptides containing DNPS-labeled tryptophans were isolated and the sequences were identified by amino acid sequence analysis and mass spectroscopy. One peptide corresponded to residues 172-176, and included Trp174. The other corresponded to the C-terminal sequence, including Trp336. It was concluded that Trp174 was at the active site of the human enzyme because it was protected by the competitive inhibitor tartrate in the DNPS-Cl modification studies. This is also consistent with the location of a homologous residue in the structure of the rat enzyme. Using site-directed mutagenesis, Trp174 was replaced by Phe or Leu. Both mutants showed altered kinetic properties, including lower Km values with several aromatic substrates, and also exhibited reduced stability towards urea denaturation.

Earlier covalent modification experiments indicated that histidine, arginine, and carboxylic acid residues were involved in the catalytic mechanism of the acid phosphatase enzyme from human prostate. The present study utilizes site-directed mutagenesis to evaluate the catalytic importance of 7 residues of human prostatic phosphatase, namely His12, His257, Asp258, Arg11, Arg15, Arg54, and Arg79, which are highly conserved in the sequences of high molecular weight acid phosphatases. Both wild type and mutant enzymes were expressed in the yeast Saccharomyces cerevisiae as fusions to a leader peptide of prepro-alpha-factor, which resulted in the secretion of processed proteins into the culture media. The kinetic parameters of the recombinant wild type enzyme were shown to be effectively identical with those of the native protein. The circular dichroism spectra of wild type and mutant proteins were effectively superimposable. Replacement of His12, which was previously shown to be transiently phosphorylated during catalysis, as well as the adjacent Arg11, both resulted in > 10(4) reductions of enzyme activity. Consistent with a postulated involvement of arginine residues in substrate binding, large reductions in Vmax caused by the R15A and R79A mutations were accompanied by marked increases in Km. However, the mutation R54A had only small effects on kinetic parameters. Essential roles for both His257 and Asp258 were also established. The D258A mutation not only caused larger decreases of Vmax toward p-nitrophenyl phosphate compared to the effect of the H257A mutation, but it also caused the reaction to become more sensitive to the nature of the leaving group. It is concluded that His12 and Asp258 are involved in the formation of the phosphoenzyme intermediate as an acceptor of the phospho group and a proton donor for the substrate leaving group, respectively, while His257 may participate in substrate binding or may facilitate the breakdown of the phosphoenzyme by maintaining the protonated state of the phosphorylated His12 residue.

Site-directed mutagenesis was used to explore the role of potential proton donors in the mechanism of the Escherichia coli acid phosphatase that is encoded by the appA gene. Asp304 appeared to be the only carboxylic acid residue that is conserved in the protein sequences of the high molecular weight acid phosphatases. The mutations Asp304Ala and Asp304Glu were introduced into appA and the corresponding proteins were overexpressed in E. coli and purified to homogeneity. Only small decreases were observed for the Km values of the substrates p-nitrophenyl phosphate, fructose 1,6-diphosphate, and tripolyphosphate. However, Vmax was greatly decreased, and the magnitude of effect depended markedly on substrate. Both mutant proteins exhibited significantly lower Vmax values with fructose 1,6-diphosphate, which possesses a much poorer leaving group than do the other two substrates. The importance of the leaving group was further tested by using a number of phenyl and alkyl phosphate derivatives as substrates. A linear correlation was observed between log Vmax and the pKa of the substrate leaving group for catalysis by the Asp304Ala mutant enzyme. These results are consistent with partition experiments using ethylene glycol as an alternate nucleophile, which indicated that for the Asp304Ala protein, the formation of a phosphoenzyme intermediate is the rate-determining step, in contrast to the situation for the wild type enzyme and the His303Ala mutant. In the latter case, the rate-limiting step of the reaction is interpreted to be the breakdown of phosphoenzyme. It is concluded that Asp304, rather than His303, is involved in protonation of the substrate leaving group.

Site-directed mutagenesis was used to examine the catalytic importance of 2 histidine and 4 arginine residues in Escherichia coli periplasmic acid phosphatase (EcAP). The residues that were selected as targets for mutagenesis were those that were also conserved in a number of high molecular weight acid phosphatases from eukaryotic organisms, including human prostatic and lysosomal acid phosphatases. Both wild type EcAP and mutant proteins were overproduced in E. coli using an expression system based on the T7 RNA polymerase promoter, and the proteins were purified to homogeneity. Examination of the purified mutant proteins by circular dichroism and proton NMR spectroscopy revealed no significant conformational changes. The replacement of Arg16 and His17 residues that were localized in a conserved N-terminal RHGXRXP motif resulted in the complete elimination of EcAP enzymatic activity. Critical roles for Arg20, Arg92, and His303 were also established because the corresponding mutant proteins exhibited residual activities that were not higher than 0.4% of that of wild type enzyme. In contrast, the replacement of Arg63 did not cause a significant alteration of the kinetic parameters. The results are in agreement with a previously postulated distant relationship between acid phosphatases, phosphoglycerate mutases, and fructose-2,6-bisphosphatase. These and earlier results are also consistent with the conclusion that 2 histidine residues participate in the catalytic mechanism of acid phosphatases, with His17 playing the role of a nucleophilic acceptor of the phospho group, whereas His303 may act as a proton donor to the alcohol or phenol.

We have partially purified an 18-kDa cytoplasmic protein from 3T3-L1 cells, which dephosphorylates pNPP and the phosphorylated adipocyte lipid binding protein (ALBP), and have identified it by virtue of kinetic and immunological criteria as an acid phosphatase (EC 3.1.3.2). The cytoplasmic acid phosphatase was inactivated by phenylarsine oxide (PAO) (Kinact = 10 microM), and the inactivation could be reversed by the dithiol, 2,3-dimercaptopropanol (Kreact = 23 microM), but not the monothiol, 2-mercaptoethanol. Cloning of the human adipocyte acid phosphatase revealed that two isoforms exist, termed HAAP alpha and HAAP beta (human adipocyte acid phosphatase), which are distinguished by a 34-amino acid isoform-specific domain. Sequence analysis shows HAAP alpha and HAAP beta share 74% and 90% identity with the bovine liver acid phosphatase, respectively, and 99% identity with both isoenzymes of the human red cell acid phosphatase but no sequence similarity to the protein tyrosine phosphatases (EC 3.1.3.48). HAAP beta has been cloned into Escherichia coli, expressed, and purified as a glutathione S-transferase fusion protein. Recombinant HAAP beta was shown to dephosphorylate pNPP and phosphoALBP and to be inactivated by PAO and inhibited by vanadate (Ki = 17 microM). These results describe the adipocyte acid phosphatase as a cytoplasmic enzyme containing conformationally vicinal cysteine residues with properties that suggest it may dephosphorylate tyrosyl phosphorylated cellular proteins.

Low molecular weight phosphotyrosyl protein phosphatases of human placenta and human red cell were purified and sequenced by a combination of Edman degradation and tandem mass spectrometry. Screening of a human placental lambda gt11 cDNA library yielded overlapping cDNA clones coding for two distinct human cytoplasmic low molecular weight phosphotyrosyl protein phosphatases (HCPTPs). The two longest clones, designated HCPTP1-1 and HCPTP2-1, were found to have identical nucleotide sequences, with the exception of a 108-base pair segment in the middle of the open reading frame. Polymerase chain reaction studies with human genomic DNA suggest that the difference between HCPTP1-1 and HCPTP2-1 does not result from alternative RNA splicing. Studies with a human chromosome 2-specific library confirmed that these sequences are located on chromosome 2, which is known to be the location of red cell acid phosphatase locus ACP1. The coding sequences of HCPTP1-1 and HCPTP2-1 were placed downstream from a bacteriophage T7 promoter and the proteins were expressed in Escherichia coli. The resulting recombinant enzymes (designated HCPTP-A and HCPTP-B, respectively) showed molecular weights of 18,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and both of them exhibited immunoreactivity with antisera raised against authentic human placental and bovine heart enzymes. The expressed proteins were highly active towards the phosphatase substrates p-nitrophenyl phosphate, beta-naphthyl phosphate, and O-phospho-L-tyrosine, but not alpha-naphthyl phosphate, threonine phosphate, or O-phospho-L-serine. HCPTP-A and -B possessed effectively identical amino acid compositions, immunoreactivities, inhibition by formaldehyde, and kinetic properties when compared with two human red cell acid phosphatase isoenzymes. It is concluded that HCPTP-A and -B are the fast and slow forms of red cell acid phosphatase, respectively, and that this enzyme is not unique to the red cell but is instead expressed in all human tissues.

An acid phosphatase (orthophosphoric monoester phosphohydrolase, acid optimum; EC 3.1.3.2) isoenzyme from wheat germ was purified 7000-fold to homogeneity. The effect of wheat germ sources and their relationship to the isoenzyme content and purification behavior of acid phosphatases was investigated. Extensive information about the purification and stabilization of the enzyme is provided. The instability of isoenzymes in the latter stages of purification appeared to be the result of surface inactivation together with a sensitivity to dilution that could be partially offset by addition of Triton X-100 during chromatographic procedures. Added sulfhydryl protecting reagents had no effect on activity or stability, which was greatest in the pH range 4-7. The purified isoenzyme was homogeneous by polyacrylamide gel electrophoresis and exhibited the highest specific activity and turnover number reported for any acid phosphatase. The molecular weights of the pure isoenzyme and of related isoenzymes from wheat germ were found to be identical (58,000). The pure isoenzyme contained a single polypeptide chain and had a negligible carbohydrate content. The amino acid composition was determined. Of the various reasons that were considered to explain isoenzyme occurrence, a genetic basis was considered most likely. The enzyme was found to exhibit substrate inhibition with some substrates below pH 6, while above pH 8 it exhibited downwardly curving Lineweaver-Burk plots of the type that are generally described as "substrate activation". The observation of a phosphotransferase activity was consistent with the formation of a covalent phosphoenzyme intermediate, while inactivation by diethyl pyrocarbonate was consistent with the presence of an active site histidine.

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.

The pairing of the half-cysteine residues of human prostatic acid phosphatase was established by proteolytic digestion and analysis of the resulting peptide mixtures by fast atom bombardment mass spectrometry (FAB-MS). An independently derived, full length cDNA clone was used as the basis for the interpretation of the FAB-MS data. The sequence of the native protein is that predicted from the present cDNA sequence, except for the carboxyl-terminal end and some possible post-translational deamidations. Isolated human prostatic acid phosphatase was found to have multiple carboxyl-terminal ends, terminating in Thr, Glu, and Asp, corresponding to residues 349-351 of the 354-residue protein that is predicted from the cDNA sequence after removal of a leader peptide. The protein contains no free sulfhydryl groups. The identical monomer chains of the dimeric native enzyme are found to contain three disulfide bonds, specifically Cys-129 to Cys-340, Cys-183 to Cys-281, and Cys-315 to Cys-319. In view of the conserved positions of cysteines in the homologous human and rat liver lysosomal acid phosphatases, an identical disulfide bonding pattern may be predicted for those proteins. The location of a potential antigenic site was established by selective labeling of proximate tyrosine residues predicted to be on the surface. A conserved RHGXRXP sequence is present in the prostatic, lysosomal, Escherichia coli, and yeast acid phosphatases and is predicted to be of mechanistic significance. In addition, residue Arg-54 is shown to be an active site residue by reaction of the enzyme with phenylglyoxal. Interestingly, this residue is present in a sequence RXRY (R,H) that is also present in lysosomal phosphatase and in recently described protein tyrosine phosphatases.

Starting with human prostatic acid phosphatase (HPAP) purified by affinity chromatography on Sepharose-bound N-(6-aminohexyl)tartramic acid [Van Etten, R. L. and Saini, M. S. (1978) Clin. Chem. 24, 1525], two forms of the enzyme can be separated by gradient elution DEAE-cellulose chromatography. The two enzyme forms are electrophoretically distinguishable on high pH gels. Under some conditions of storage one of these forms undergoes a conversion to the other. Both forms have identical amino acid compositions and Vmax values with typical substrates, but differences are observed in circular dichroism spectra, relative fluorescence intensities, Km values, and carbohydrate composition. It is concluded that the two forms differ only in the structure of their (nonphosphorylated) carbohydrate side chains. In view of the possible interconversion of the two forms upon storage, caution must be observed in any attempt to attach clinical significance to the relative amounts of the two forms.

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.

Treatment of homogenous human prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) with low concentrations of Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) leads to a rapid loss of enzymic activity. The rate of inactivation of the enzyme is reduced in the presence of the competitive inhibitors phosphate and L-(+)-tartrate, but not in the presence of non-inhibitory D-tartrate. Measurement of the ethylamine produced upon hydrolysis of enzyme modified in the presence of D- and of L-tartrate permitted the quantitative estimation of the number of carboxylic acid residues at the active site. The data indicate that two carboxyl groups per (dimeric) enzyme molecule are essential for catalytic activity. It is proposed that one function of the active site carboxyl group may be to protonate the leaving alcohol or phenol portion of the phosphomonoester substrate during the formation of the covalent phosphoenzyme intermediate.

We describe the synthesis of a long-chain monoamide derivative of L(+)-tartaric acid and its attachment to Sepharose 4B. Procedures are then described for use of this material in purifying human prostatic and (for the first time) canine prostatic acid phosphatases to constant specific activity and electrophoretic homogeneity. Depending on sample size, such purification is possible in one step, and is clearly faster and more efficient than are previously described methods. These materials and procedures have significant potential in studies of the comparative biochemistry and clinical chemistry of tartrateinhibitable acid phosphatases.

A 17-kilodalton (kDa) human placental acid phosphatase was purified 21,400-fold to homogeneity. The enzyme has an isoelectric point of pH 7.2 and a specific activity of 106 mumol min-1 mg-1 using p-nitrophenyl phosphate as a substrate at pH 5 and 37 degrees C. This placental acid phosphatase showed activity toward phosphotyrosine and toward phosphotyrosyl proteins. The pH optima of the enzyme with phosphotyrosine and with phosphotyrosyl band 3 (from human red cells) were between pH 5 and 6 and pH 5 and 7, respectively. The Km for phosphotyrosine was 1.6 mM at pH 5 and 37 degrees C. Phosphotyrosine phosphatase activity was not inhibited by tartrate or fluoride, but vanadate, molybdate, and zinc ions acted as strong inhibitors. Enzyme activity was also inhibited by DNA, but RNA was not inhibitory. It is a hydrophobic nonglycoprotein containing approximately 20% hydrophobic amino acids. The average hydrophobicity was calculated to be 903 cal/mol. The absorption coefficient at 280 nm, E1% 1cm, was determined to be 5.7. The optical ellipticity of the enzyme at 222 nm was -5200 deg cm2 dmol-1, which would correspond to a low helical content. Free sulfhydryl and histidine residues were necessary for the enzyme activity. The enzyme contained four reactive sulfhydryl groups. Chemical modification of the sulfhydryls with iodoacetate resulted in unfolding of the protein molecule as detected by fluorescence emission spectroscopy. Antisera against both the native and the denatured protein were able to immunoprecipitate the native enzyme. However, upon denaturation, the acid phosphatase lost about 70% of the antigenic determinants. Both antisera cross-reacted with a single 17-kDa polypeptide on immunoblotting.

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.

Human prostatic acid phosphatase is known to display considerable charge heterogeneity upon isoelectric focusing. The structural basis of this heterogeneity is not known, although it has been widely attributed to variations in the nature of the carbohydrate chains or to substituents on the carbohydrate chains of the glycoprotein. In this study, the role of the carbohydrate chains in the charge heterogeneity of the protein was examined. First, sialic acid residues were removed by treatment of the acid phosphatase with neuraminidase. The desialo enzyme was fractionated and purified by L-tartramic acid affinity chromatography. Then, after the protein oligosaccharide linkages were made accessible by the presence of NP-40 or by denaturing the protein, the protein was completely deglycosylated by endo-beta-N-acetylglucosaminidase F at pH 4.5 and 9.3. Two discrete intermediates were clearly resolved by SDS gel electrophoresis during the deglycosylation of the denatured protein at pH 9.3, indicating the existence of three sites of glycosylation on the protein. Peptide mixtures were obtained by digestion of carboxymethylated and citraconylated derivatives of the enzyme with trypsin and the glycopeptides were isolated. The amino acid compositions of the glycopeptides were consistent with the interpretation that there are a minimum of two sites of glycosylation on each peptide subunit of the enzyme. Isoelectric focusing experiments on the native, desialo, and denatured, deglycoso acid phosphatase showed that the heterogeneity of the protein is not eliminated either by desialylation or by deglycosylation. Thus, the electrophoretic heterogeneity of human prostatic acid phosphatase does not lie primarily in the oligosaccharide part of the glycoprotein or in altered conformational states of the protein, but in structural variations of the polypeptide itself. The heterogeneity may be due to variations at the C-terminus, partial deamidation, phosphorylation, sulfation or other posttranslational modifications of the protein chain.

The stereochemical course of the phospho transfer catalyzed by homogeneous human prostatic acid phosphatase was investigated using 31P nuclear magnetic resonance spectroscopy. Transphosphorylation from phenyl-(R)-[15O, 17O, 18O]phosphate to (S)-propane-1,2-diol occurs with overall retention of configuration at phosphorus. This stereochemical result is consistent with the interpretation that the hydrolysis of substrates by this enzyme proceeds by way of a covalent phosphoenzyme intermediate. Conditions for optimizing phospho transfer by this and related acid phosphatases have also been explored.

The biosynthesis of distinct prostatic and lysosomal acid phosphatases is demonstrated using a human prostatic carcinoma cell line, PC-3SF12. The biosynthesis and maturation of the acid phosphatases was studied by metabolic labeling with radioactive leucine, specific immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. Of the tartrate-inhibitable acid phosphatase activity in PC-3SF12 cells, 60% is lysosomal and 10% is prostatic. The lysosomal-type acid phosphatase is synthesized as precursor with a molecular weight of 68,000, some of which is converted to higher-molecular-weight precursor polypeptides (Mr 71,000 and 77,000). The multiple forms of the precursors are due to differences in the carbohydrate chains on the enzyme because biosynthesis in the presence of tunicamycin eliminates the precursor multiplicity. The initial precursor (Mr 68,000) is processed to a mature polypeptide (Mr 49,000), via intermediates with molecular weights of 62,000 and 59,000. The mature polypeptide is degraded to smaller polypeptides with molecular weights of 30,000, 28,000, and 25,000. Precursor polypeptides of the lysosomal-type enzyme are secreted in the medium. Prostatic acid phosphatase is synthesized as a precursor with a molecular weight of 110,000, which is processed via several intermediates (Mr 99,000-93,000, 77,000, and 55,000) to a mature polypeptide with a molecular weight of 49,000. Particularly during cell homogenization, or lysis, the mature polypeptide is rapidly degraded to an immunoprecipitable polypeptide with a molecular weight of 20,000. None of these polypeptides is secreted in detectable amounts into the medium. Precursors and mature and smaller polypeptides are present in human prostate extract and seminal fluid. Proteolytic degradation of prostatic acid phosphatases in cells and tissues is probably catalyzed by a plasmin-like or related trypsin-like enzyme because degradation of the mature prostatic phosphatase polypeptide is completely prevented by addition of the plasmin inhibitor bovine pancreatic trypsin inhibitor. Prostatic- and lysosomal-type acid phosphatases are eventually stored at least in part in two different types of cell organelles. Testosterone does not influence the biosynthesis and secretion of either acid phosphatase in this cell line.

Human prostatic-type of acid phosphatase has been demonstrated by biochemical methods to be expressed in a number of cells and tissues in addition to the prostate gland. However, the function of this activity is unknown, nor has the enzyme been convincingly localized at the cellular level in any non-prostatic tissues. Using biochemical and immunocytochemical methods, we demonstrate that human intestinal epithelium contains both a lysosomal and prostatic type of acid phosphatase. The prostatic-type enzyme is present only in the epithelium of the crypts and to a lesser extent in the transitional zone at the base of the villi, in contrast to the widely-distributed lysosomal type. The prostatic enzyme is contained in granules that do not react with anti-lysosomal acid phosphatase and are probably secretory in nature.

The biosynthesis of the prostatic form of human acid phosphatase was studied in normal embryonic lung cells, WI-38, by metabolic labeling with tritiated leucine and [32P]phosphate, followed by specific immunoprecipitation, gel electrophoresis, and fluorography. Of the total tartrate-inhibitable acid phosphatase activity in WI-38 cells, 30% is due to the prostatic form. The primary translation product that leads eventually to the mature prostatic enzyme is a precursor polypeptide of 112 kDa. The precursor polypeptide is processed to mature polypeptides of 59, 55, and 49 kDa via an intermediate 91-kDa precursor. WI-38 cells also secrete a 113-kDa peptide into the medium. The precursor and mature polypeptides are glycosylated and phosphorylated. Upon treatment with endo-beta-hexosaminidase H, the apparent molecular weighs of the polypeptides are reduced by approximately 4 kDa and phosphate is lost.

The biosynthesis of lysosomal acid phosphatase was studied in a normal human embryonic lung cell line, WI-38. Cells were labeled with radioactive leucine under a variety of conditions, the enzyme was immunoprecipitated using a monospecific antiserum raised against human liver lysosomal acid phosphatase, and the products were separated by electrophoresis and were visualized by fluorography. Lysosomal acid phosphatase constitutes 60% of the total tartrate-inhibitable acid phosphatase in WI-38. It is initially synthesized as a high-molecular-weight precursor polypeptide of 69 kDa. The precursor polypeptide is rapidly glycosylated and processed to a mature enzyme of 53-45 kDa via intermediates of 65 and 60 kDa in WI-38 cells. The 69-kDa precursor polypeptide is also converted to larger precursor polypeptides of 74 and 80 kDa. The multiplicity of precursor polypeptides is due at least in part to differences in the glycosylation and phosphorylation of the polypeptides. Sensitivity of phosphorylated oligosaccharide chains from precursor, mature and small polypeptides to endo-beta-hexosaminidase H-catalyzed cleavage suggests the presence of high-mannose phosphorylated oligosaccharide chains similar to those present on many other lysosomal enzymes. The effects of tunicamycin and ammonium chloride were also studied. In contrast to the effect of ammonium chloride on arylsulfatase A secretion, the lysosomal acid phosphatase in WI-38 cells was not secreted in the presence of NH4Cl. This is consistent with the existence of an alternate route for the transfer of lysosomal acid phosphatase into lysosomes. This alternate route may be the reason that I-cell fibroblasts contain a normal level of lysosomal acid phosphatase.

The structures of the oligosaccharides comprising the carbohydrate moieties of human prostatic acid phosphatase were elucidated by 1H NMR spectroscopy. Homogeneous enzyme was digested with Pronase P, and three asparagine-linked carbohydrate moieties were obtained upon fractionation of the digest using a concanavalin A-Sepharose affinity column. One fraction did not bind to the column, while the portion that did bind was separated into two fractions by elution with two concentrations of mannose. The high-resolution 1H NMR spectra for the three fractions were recorded at 470 MHz. From these data, the structures were deduced to be high mannose, partially sialylated and fucosylated biantennary complex, and fucosylated, partially sialylated triantennary complex oligosaccharides. No O-linked carbohydrate moiety was detected, although the possible presence of small O-linked oligosaccharides cannot be completely discounted from these data.

We studied possible causes of the electrophoretic heterogeneity of the acid phosphatase (EC 3.1.3.2) purified by affinity chromatography from human prostate and human seminal fluid. The isoelectric focusing pattern in polyacrylamide gel shows numerous bands in the pH range 4.0-5.2 and 5.5-5.9. Treatment with neuraminidase under conditions shown to cause complete removal of sialic acid does not abolish the observed heterogeneity. Although there is a change of the more acidic forms to ones having more basic pI values, at least 4 distinct bands remain. Structural differences at the amino terminal end can be ruled out as the cause of the remaining electrophoretic heterogeneity. Lysine is shown to be the amino terminal amino acid for both the prostatic and seminal fluid enzymes. The sequences of the first 23 amino acids are shown to be identical for the prostatic and seminal fluid acid phosphatases. The functional enzyme contains no metal ion but it can be stoichiometrically inactivated by cupric ion.

A homogeneous, tartrate-inhibitable acid phosphatase (AcPase) was obtained from the liver of channel catfish (Ictalurus punctatus) by the use of Affi Gel-10-coupled aminohexyltartramic acid affinity chromatography. The enzyme has a molecular weight of 82,500 and is a dimer consisting of two apparently equivalent subunits with subunit weights of 35,000 +/- 3000. Amino acid composition data are presented and compared with those of mammalian acid phosphatases. Data suggest that the enzyme is a metalloacid phosphatase. Catfish liver AcPase exhibits two molecular forms with pI 5.66 and 5.37 which were separated by chromatofocusing. A spontaneous conversion of the less acidic form to a more acidic form was observed and this conversion was accompanied by a decreased sensitivity towards tartrate inhibition.

Three different antisera against human prostatic acid phosphatase were used for direct and indirect immunohistochemical demonstration of acid phosphatase in paraffin sections of infantile and adult normal, hyperplastic and carcinomatous prostatic tissue. All antisera were prepared in rabbits. Antiserum A was prepared from highly purified acid phosphatase extracted from autopsy specimens. Antiserum B was a concentrate of a commercial antiserum used in radioimmunoassay and was prepared from purified extracts of human seminal fluid. Antiserum C was a peroxidase-conjugated antiserum prepared from purified extracts of human seminal fluid. The specificity of the three antisera was compared using different immunohistochemical methods and tissues. It was comparably high in all three antisera which gave only slightly different staining results in prostatic tissue. The staining results in prostatic carcinoma were only dependent on the titer of the respective antiserum. Carcinomas with a cribriform growth pattern showed variable staining, but always had a positive immunoreactions, provided the titer of the antiserum was sufficiently high. Striking differences were observed in metaplastic, atrophic and hyperplastic prostatic epithelium. The most intense reaction was observed in atrophic glands: it was much less intense in hyperplastic and normal epithelium and negative or slightly positive in metaplastic epithelium.

Two forms of catfish liver acid phosphatase (AcPase I and II) were separated and purified to homogeneity and their carbohydrate compositions and some biochemical properties were studied. Evidence is given that AcPase I and II are differently glycosylated forms of the same enzyme. The enzyme forms differ significantly in the size and the composition of their carbohydrate components, sensitivity towards sulfhydryl-blocking and protecting reagents, sensitivity to ferric and ferrous ions, thermostability and ability to hydrolyze some nucleotides. The more highly glycosylated form is more sensitive to thermal denaturation. AcPase I and II behave differently towards ascorbate and changes in its concentration and it is suggested that the concentration of reducing modifiers may regulate AcPase activity at the cellular level. It is hypothesized that the differing extents of glycosylation influence the structure of the enzyme forms. This is expressed in altered conformations of two enzyme forms and results in a different exposure of the essential cysteine residues.

Treatment of human prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) with either of the arginine-specific modifiers 2,3-butanedione or 1,2-cyclohexanedione in borate buffer at pH 8.1 leads to loss of activity. The inactivation by cyclohexanedione can be partially reversed by 0.2 M hydroxylamine. The rate of inactivation by both modifiers is decreased in the presence of the competitive inhibitors L-(+)-tartrate or inorganic phosphate but not in the presence of the non-inhibitor D-(-)-tartrate. Amino acid analysis of modified acid phosphatase indicates that only arginines are modified and that L-(+)-tartrate protects at least two arginyl residues from modification. A likely role of these arginyl residues is their involvement in binding the negatively charged phosphate group of the substrate.

The hydrolysis of phosphoric monoesters by acid phosphatases is thought to proceed via the formation of a phosphoenzyme intermediate, but no stereochemical evidence exists on this point. We have carried out the transphosphorylation from phenyl (R)-[16O, 17O, 18O] phosphate to (S)-propane-1,2-diol in the presence of homogeneous bovine liver acid phosphatase, and have found that the reaction proceeds with greater 90% overall retention of configuration at phosphorus. This stereochemical course of phospho transfer during the enzyme-catalyzed reaction of the phosphoric monoester is consistent with a double displacement mechanism in which each step proceeds with inversion of the stereochemistry at phosphorus, and is similar to the behavior of the bacterial alkaline phosphatase.

Human liver contains three chromatographically distinct forms of non-specific acid phosphatase (EC 3.1.3.2). Acid phosphatases I, II and III have molecular weights of greater than 200 000, of 107 000, and of 13 400, respectively. Following partial purification, isoenzyme II was obtained as a single activity band, as assessed by activity staining with p-nitrophenyl phosphate and alpha-naphthyl phosphate on polyacrylamide gels run at several pH values. With 50mM p-nitrophenyl phosphate as a substrate, enzymes II and III exhibit plateaus of activity over the pH range 3 - 5 and 3.5 - 6, respectively. Acid phosphatase II is not significantly inhibited by 0.5% formaldehyde. The activity of human liver acid phosphatase II and of human prostatic acid phosphatase towards several substrates is compared. The liver enzyme, is marked contrast to the prostatic enzyme, does not hydrolyze O-phosphoryl choline.

Conclusive evidence is presented that an acid phosphatase catalyzes phosphate (oxygen)-water exchange. Studies conducted with human prostatic acid phosphatase by two independent methods have established that, despite earlier reports to the contrary, the enzyme catalyzes an exchange reaction between oxygen atoms of phosphate ion and of water. Kinetic data were obtained both by chemical conversion to trimethyl phosphate followed by mass spectroscopy and by a totally independent method involving 31P isotope shift nuclear magnetic resonance spectroscopy. Analysis showed that the enzyme catalyzes the exchange in a random, noncoupled process. If any coupled exchange occurs, it must represent less than 10% of the total. By mass spectral analysis, catalytic rate constants kcat = 0.14 sec-1 (4 degrees) and 1.8 sec-1 (37.5 degrees) were obtained. By 31P nuclear magnetic resonance kcat = 1.6 sec-1 (31 degrees) was obtained. The energy of activation for the exchange reaction is approximately 13kcal mol-1. The kcat value for exchange is about 10-fold greater than that observed with Escherichia coli alkaline phosphatase.

We describe an assay method using choline O-phosphate as a substrate for the measurement of serum prostatic acid phosphatase as an aid in the diagnosis of prostatic cancer. Choline phosphate is hydrolyzed by homogeneous prostatic acid phosphatase, and it is also hydrolyzed by an acid phosphatase present in the serum of prostatic carcinoma patients. In contrast, serum samples from apparently healthy persons do not exhibit any significant choline O-phosphate phosphatase activity. There is a correlation of 98% (n = 46) between choline O-phosphate phosphatase activity and typical measurement for prostatic acid phosphatase activity carried out using thymolphthalein monophosphate as the substrate. The new method appears to be as accurate as colorimetric methods based on thymolphthalein phosphate as a substrate. Although not as sensitive as immunologically based methods, the present technique for measuring prostatic acid phosphatase activity using choline phosphate as a substrate is economical and relatively simple.

Human prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) is a dimeric (alpha 2) protein that catalyses the hydrolysis of phosphomonoesters. Several reports suggest that a phosphoenzyme intermediate is involved in the mechanism of acid phosphatase. Chemical modification studies and trapping experiments were therefore undertaken in order to ascertain the identity of the amino acid residue(s) involved in the formation of this intermediate. Human prostatic acid phosphatase is inactivated by diethyl pyrocarbonate (second-order rate constant of 7 M-1. min-1 at pH 6.2) with an accompanying increase in absorbance at 242 nm due to formation of ethoxyformylhistidyl derivatives. In the presence of competive inhibitors the rate of inactivation is decreased. Inactivation can be partially reversed by hydroxylamine. The pH curve of inactivation indicates the involvement of a residue having a pK alpha of 6.5. Direct evidence for the involvement of a histidine residue in the mechanism was obtained by trapping a covalent phosphohistidyl-enzyme intermediate. Incubation of the enzyme with p-nitrophenyl [32 P] phosphate leads to incorporation of 0.44 mol 32P/mol enzyme. The denatured phosphoenzyme,which was acid labile but base stable, was hydrolyzed in 3 M KOH and the radioactivity was found to cochromatograph with synthetic tau-phosphohistidine on Dowex-1 ion-exchange resin. These results are consistent with a catalytic mechanism involving histidine as a nucleophile in the formation of a covalents phosphoenzyme intermediate.

The pH dependence of the human prostatic acid phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate and beta-glyceryl phosphate has been studied over a wide range of pH and the values of Km and V calculated with the aid of the Cleland HYPER program. The pH dependence of Km shows the effect of substrate ionization: pK values of 5.6 and 6.4 are observed as for the respective values of free substrates. The pH dependence of both Km and V for each substrate reveals the involvement of an ionizable group in the ES complex which is ascribed to a phosphohistidine-enzyme intermediate. The small deuterium solvent isotope effects which are observed on V are consistent with values observed for solvolysis of phosphoramidates. The measured data for Km indicates limits on burst-titration experiments of prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2).

A simple, rapid and efficient procedure is presented for the purification of human prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) to homogeneity. The method employs two steps suitable for use with large quantities of material, followed by chromatography on concanavalin A-Sepharose as its sole column step. The procedure also permits the recovery of purified enzyme in higher yields than earlier methods.

Two isoenzymes of rat liver acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum) EC 3.1.3.2) have been purified to homogeneity, at least one of these for the first time. Both of the rat liver isoenzymes have identical specific activities towards p-nitrophenyl phosphate. Molecular weights of the native enzymes are 92 000 for rat liver isoenzyme I and 93 000 for isoenzyme II, while the subunit molecular weights are 51 000 and 52 000 respectively. Data on substrate specificity and pH dependence are presented for the homogeneous canine prostatic enzyme, which is also isolated as a dimeric enzyme of (native) molecular weight 89 000. Carbohydrate analysis data are presented for canine prostatic acid phosphatase and it is further noted that both isoenzymes of rat liver acid phosphatase are also glycoproteins. The amino acid compositions of the two rat liver isoenzymes are presented together with those of the similar dimeric acid phosphatase of human liver and of canine prostate. Comparison of these results with published data for the amino acid composition of human prostatic acid phosphatase shows substantial similarities. However, significant differences are seen in the amino acid composition of rat liver acid phosphatase isoenzyme I as compared to a previous literature report. Most notably, 17 histidine residues are found per mol of isoenzyme I and 18 for isoenzyme II.

We describe the development of a stable reference material for prostatic acid phosphatase, derived from human prostatic tissue and human seminal fluid. The enzyme was purified by an L-tartramic acid affinity-chromatography technique. Two-dimensional electrophoresis revealed essentially no contaminating proteins, and specific tests revealed no contaminating enzymes. The preparations, in a matrix containing 30 g of human serum albumin and 0.1 mol of sodium acetate per liter, pH 6.0, were studied with respect to stability of both catalytic activity and immunological identity. We conclude that the preparations from either source are satisfactorily stable, and that either is acceptable for use in preparing clinical reference materials. These materials will be used in developing a reference method.