Hydrolysis of the brain dipeptide N-acetyl-L-aspartyl-L-glutamate. Identification and characterization of a novel N-acetylated alpha-linked acidic dipeptidase activity from rat brain

High performance liquid chromatography studies documented the presence of an enzyme activity, N-acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase), in rat brain membranes that cleaves the endogenous brain dipeptide, N-acetyl-L-aspartyl-L-glutamate to N-acetyl-aspartate and glutamate. With ion exchange chromatography, which quantitatively separated [3,4-3H]glutamate from N-acetyl-L-aspartyl-L-[3,4-3H]glutamate, we found that NAALA dipeptidase activity was essentially restricted to nervous tissue and kidney. We characterized NAALA dipeptidase activity in lysed synaptosomal membranes obtained from rat forebrain. Membrane-bound NAALA dipeptidase activity was optimal between pH 6.0 and 7.4 at 37 degrees C. Eadie-Hofstee analysis of kinetic data revealed a rather high apparent affinity for N-acetyl-L-aspartyl-L-glutamate with a Km = 540 nM and a Vmax = 180 nM/mg of protein/min. While NAALA dipeptidase showed a requirement for monovalent anions such as Cl-, the polyvalent anions phosphate and sulfate inhibited enzyme activity 50% at 100 microM and 1 mM, respectively. The divalent metal ion chelators EGTA, EDTA, and o-phenanthroline completely abolished activity, which was partially restored by manganese. Treatment of membranes with 1 mM dithiothreitol abolished NAALA dipeptidase activity. NAALA dipeptidase activity was also sensitive to the aminopeptidase inhibitors bestatin and puromycin, although not to the selective aminopeptidase A inhibitor amastatin. Structure-activity relationships inferred from inhibitor studies suggest that this enzyme shows specificity for N-acetylated alpha-linked acidic dipeptides. NAALA dipeptidase was also potently inhibited by the excitatory amino acid agonist L-quisqualate. Comparison of the properties of NAALA dipeptidase to those of previously characterized enzymes suggests that this is a novel peptidase which may be involved in the synaptic degradation of N-acetyl-L-aspartyl-L-glutamate.     

Purification and characterization of human microsomal dipeptidase

Human microsomal dipeptidase (MDP, formerly referred to as dehydropeptidase-I or renal dipeptidase) [EC 3.4.13.11] was solubilized from the membrane fraction of kidney by treatment with octyl-beta-D-glucoside and purified by a procedure including ion exchange chromatography and affinity chromatography on cilastatin-immobilized Sepharose. The purified human MDP was found to be homogeneous on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The apparent molecular weight (Mr) was estimated by SDS-polyacrylamide gel electrophoresis under non-reducing conditions to be 130 kDa, comprising a homodimer of two subunits. After treatment with endoglycosidase F, human MDP showed a single band with an apparent Mr of 42 kDa on SDS-polyacrylamide gel electrophoresis. Human MDP was found to bind to Con A-Sepharose and the activity was eluted with methyl-alpha-D-mannopyranoside, suggesting that human MDP is a glycoprotein. We also examined the substrate specificity of human MDP and found that human MDP catalyzed the hydrolysis of S(substituent)-L-cysteinyl-glycine adducts such as L-cystinyl-bis(glycine) and S-N-ethylmaleimide-L-cysteinyl-glycine, as well as the conversion of leukotriene D4 to leukotriene E4. These results suggest that MDP might play an important role in the metabolism of glutathione and leukotriene.     

Post-proline dipeptidyl-aminopeptidase from synaptosomal membranes of guinea-pig brain. A possible role for this activity in the hydrolysis of His-ProNH2, arising from the action of synaptosomal membrane pyroglutamate aminopeptidase on thyroliberin

In this paper we report that while 55% of the total post-proline dipeptidyl-aminopeptidase activity in guinea-pig brain is associated with the soluble fraction of the cells, the remaining activity is widely distributed throughout the particulate fractions. A significant portion of this particulate activity is, however, associated with a synaptosomal membrane fraction. The specific activity of this enzyme rose as the synaptosomal membrane fraction was prepared from a synaptosomal fraction and had previously risen at the synaptosomal fraction was prepared from a postmitochondrial pellet. The synaptosomal membrane post-proline dipeptidyl-aminopeptidase was released from the membrane by treatment with Triton X-100 and partially purified by chromatography on Sephadex G-200. By contrast with the soluble enzyme the partially purified solubilised synaptosomal membrane post-proline dipeptidyl-aminopeptidase was not inhibited by 1.0 mM p-chloromercuribenzoate, 1.0 mM N-ethylmaleimide or 0.5 mM puromycin but was inhibited by 0.5 mM bacitracin. The partially purified solubilised enzyme was capable of releasing His-Pro from His-Pro-Val, His-Pro-Leu, His-Pro-Phe and His-Pro-Tyr and of releasing Gly-Pro from Gly-Pro-Ala but could not release Arg-Pro from Arg-Pro-Pro or from Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (bradykinin). It was also unable to release Pro-Pro from Pro-Pro-Gly or Glp-Pro from Glp-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-MetNH2 (eledoisin). Using [Pro-3H]thyroliberin we show that the membrane-bound enzyme converts His-ProNH2, produced by the action of the synaptosomal membrane pyroglutamate aminopeptidase, to His-Pro thus competing with the spontaneous cyclisation of His-ProNH2 to His-Pro diketopiperazine. Purified preparations of synaptosomal membrane pyroglutamate aminopeptidase were used to generate His-ProNH2, which could then be converted to His-Pro by the presence of the partially purified synaptosomal membrane post-proline dipeptidyl-aminopeptidase. This preparation was free of contaminating post-proline cleaving endopeptidase, carboxypeptidase P, aminopeptidase P, prolyl carboxypeptidase or proline dipeptidase.     

Preparative purification of dipeptidyl peptidase IV.

Dipeptidyl-Peptidase IV was purified from pig kidney by ammonium sulfate fractionation, gel filtration, QAE-cellulose chromatography and affinity columns with Gly-Pro- and Concanavalin A-Sepharose. The specific activity of the purified enzyme is 41.8 units/mg. Polyacrylamide gel electrophoresis and silver staining show a single band. The enzyme preparation is free of aminopeptidase and dipeptidase activity, proved fluorimetrically and by gas chromatography/mass spectrometry. The most important procedure for removal of contaminating enzyme activities is a stepwise NaCl-gradient on a QAE-ZetaPrep ion exchange disk.     

Purification and Characterization of a Dipeptidase from Lactobacillus helveticus SBT 2171

A metal-dependent dipeptidase was purified to homogeneity from a cell extract of Lactobacillus helveticus SBT 2171 by fast protein liquid chromatography. The enzyme was purified 237-fold from the extract, with a yield of 1.8%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 50,000. The dipeptidase hydrolyzes a range of only dipeptides. Dipeptides containing proline, glutamic acid, and aspartic acid are not hydrolyzed. The enzyme was shown to be a metalloenzyme with a pH optimum of 8.0 and a temperature optimum of 55(deg)C. Dithiol-reducing reagents exert strong inhibition on enzyme activity. Kinetic studies indicated that the enzyme has a relative average affinity for leucyl-leucine (K(infm), 0.5 mM). The negative immunoresponse of the purified enzyme with monoclonal antibodies raised against a dipeptidase from Lactococcus lactis subsp. cremoris Wg2 shows that both enzymes can be immunologically distinguished.     

Purification and reconstitution of the high affinity choline transporter

The high-affinity choline transporter has been solubilized from synaptosomal membranes by various detergents. The solubilized carrier protein has been incorporated into liposomes after removal of the detergent by dialysis. Using the reconstitution of choline transport activity as an assay, the components catalyzing choline translocation were purified from the detergent extract by ion-exchange chromatography on a Mono-Q column followed by immunoaffinity chromatography. Monitoring the active fractions by sodium dodecylsulfate polyacrylamide gel electrophoresis and isoelectrofocussing gave one major protein with an apparent molecular weight of about 90,000 and an isoelectric point of pH 4.7. The isolated protein appeared to be heavily glycosylated as shown by lectin binding; upon treatment with endoglycosidase F the polypeptide was degraded to an apparent molecular weight of about 65,000. Accumulation of choline into liposomes reconstituted with the purified protein was driven by artificially imposed sodium gradients and inhibited by hemicholinium-3.     

Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.     

Preparative Purification of Dipeptidyl Peptidase IV

Abstract

Dipeptidyl-Peptidase IV was purified from pig kidney by ammonium sulfate fractionation, gel filtration, QAE-cellulose chromatography and affinity columns with Gly-Pro- and Concanavalin A-Sepharose. The specific activity of the purified enzyme is 41.8 units/mg. Polyacrylamide gel electrophoresis and silver staining show a single band. The enzyme preparation is free of aminopeptidase and dipeptidase activity, proved fluorimetrically and by gas chromatography/mass spectrometry. The most important procedure for removal of contaminating enzyme activities is a stepwise NaCl-gradient on a QAE-ZetaPrep ion exchange disk.     

The purification and characterization of a proline dipeptidase from guinea pig brain

A proline dipeptidase (EC 3.4.13.9) from guinea pig brain was purified to over 90% homogeneity by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, calcium phosphate-cellulose chromatography, chromatofocusing, and gel filtration on Sephadex G-200. A purification factor of 2718-fold was obtained with a yield of 7%. The purified enzyme was found to have an apparent molecular weight of 132,000 and to consist of two dissimilar subunits of molecular weights 64,000 and 68,000. The substrate specificity of the enzyme is not that of a strict proline dipeptidase. Although it preferentially hydrolyzes proline dipeptides (Leu-Pro) it also hydrolyzes prolyl dipeptides (Pro-Leu) and dipeptides not containing proline (Leu-Leu). The purified enzyme preparation exhibited weak aminoacylproline aminopeptidase activity against Arg-Pro-Pro but it did not exhibit any post-proline dipeptidyl aminopeptidase, post-proline cleaving endopeptidase, proline iminopeptidase, prolyl carboxypeptidase or carboxypeptidase P activities when tested with a large variety of peptides and arylamides. With all of the proline and prolyl dipeptides examined the enzyme exhibited biphasic kinetics (two distinct slopes on Lineweaver-Burk plots). However, with Leu-Leu as substrate normal Michaelis-Menten kinetics were obeyed.     

Purification and characterization of cholesterol sulfotransferase from rat skin.

Previous work has demonstrated that the activity of the enzyme cholesterol sulfotransferase is rapidly and dramatically increased upon squamous differentiation of a variety of epithelial cells in culture, including epidermal keratinocytes. As a step toward understanding the molecular mechanisms underlying this differentiation-related change, we now report the partial purification and characterization of this enzyme activity from rat skin. Supernatant solutions from rat skin homogenates were subjected to a series of column chromatography steps including anion exchange, gel filtration, chromatofocusing and hydrophobic interaction chromatography. The purification procedure resulted in cholesterol sulfotransferase activity purified 2,700-fold with a 11% recovery. The most purified preparation yielded a major Coomassie blue-stained band on denaturing polyacrylamide gel electrophoresis of an apparent molecular weight (MW) of 40,000 Da. Photoaffinity labeling with the donor substrate, 3'-phosphoadenosine-5'-phospho-[35S]-sulfate resulted in a single radiolabeled protein band on denaturing polyacrylamide gel electrophoresis, again of apparent MW 40,000 Da, strongly suggesting that the major Coomassie blue-stained band in the most purified preparation is the cholesterol sulfotransferase protein. Among 3beta-hydroxysteroids with a A5 double bond that were tested, each served as a substrate, while androgens, estrogens, corticosteroids, p-nitrophenol and DOPA did not serve as substrates. Apparent Michaelis constants for the 3beta-hydroxysteroid substrates ranged from 0.6 to 8 microM.     

Purification and properties of human pancreas dipeptidase

Dipeptidase [EC 3.4.13] was purified from human pancreas; the activity was followed with L-Leu-L-Leu as a substrate. Polyacrylamide gel electrophoresis showed that the final preparation was homogeneous. The molecular weight of the dipeptidase was estimated to be 135,000 by gel filtration. From the result of SDS-polyacrylamide gel electrophoresis, it was found that the enzyme consisted of two subunits with equal molecular weights of 68,000. By atomic absorption analysis, the dipeptidase was shown to be a zinc metalloenzyme containing one atom of zinc for each subunit. Cu2+ and Hg2+ (1 mM) inhibited the enzyme by 50%. o-Phenanthroline strongly inhibited the enzyme. The dipeptidase hydrolyzed dipeptides such as L-Ala-L-Ala, L-Met-L-Met, L-Ala-L-Leu, L-Leu-Gly, and L-Leu-L-Leu but did not hydrolyze tripeptides, Bz-amino acids, CBz-amino acids, or L-amino acid beta-naphthylamides. The dipeptidase from human pancreas was immunologically distinct from human liver dipeptidase.     

Purification of phosphatidylinositol kinase from bovine brain myelin.

A membrane-bound phosphatidylinositol (PI) kinase (EC 2.7.1.67) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme.     

Purification of the putative alpha-latrotoxin receptor from bovine synaptosomal membranes in an active binding form.

alpha-Latrotoxin (alpha-LTx, apparent mol. wt. 130 000) is a presynaptically active neurotoxin purified from the venom of the black widow spider that causes massive exocytotic release of neurotransmitters, presumably via binding to presynaptic membrane protein(s). Solubilization and purification experiments were undertaken to identify and characterize this membrane component. An immunoaffinity matrix was prepared by sequentially binding anti-alpha-LTx antibodies and alpha-LTx to Protein A-Sepharose CL-4B. Beads were irreversibly cross-linked with dimethyl pimelimidate. These beads were capable of extracting alpha-LTx binding activity from Triton X-100 solubilized bovine synaptosomal membranes. Following extensive washing, bound material was eluted with 6 M urea. Analysis of silver stained and radiolabel-containing gels revealed one major band (apparent mol. wt. 200 000) under non-reducing conditions and two major bands (apparent mol. wts. 66 000 and 54 000) under reducing conditions. The purified material was still capable of specifically binding alpha-LTx as determined by solid phase assays on microtiter plates. The affinity for alpha-LTx of the purified preparation was similar to that of the native membrane (KA approximately 10(10) M). It is concluded that a putative alpha-LTx receptor protein can be purified from synaptosomal membranes using an immunoaffinity matrix in a form that retains its defined biological property (alpha-LTx binding).     

Purification of the muscarinic acetylcholine receptor from porcine brain

The muscarinic acetylcholine receptor of porcine cerebrum has been purified to apparent homogeneity by affinity chromatography, with conjugated 3-(2'-aminobenzhydryloxy)tropane (ABT) as described previously (Haga, K., and Haga, T. (1983) J. Biol. Chem. 258, 13575-13579). In a single step purification using 900 ml of digitonin/cholate-solubilized preparations and 300 ml of the ABT-agarose gel, we obtained, in a yield of 10-15%, more than 250 pmol of muscarinic receptors which bind [3H]N-methylscopolamine with a specific activity of 1,000-5,000 pmol/mg of protein (1,000-5,000-fold purification). The muscarinic receptors eluted from the ABT-agarose gel with 0.1 mM atropine were adsorbed to hydroxylapatite and then recovered as a concentrated solution. Muscarinic receptors were further purified by rechromatography with the same gel or by gel permeation high pressure liquid chromatography. The amino acid composition of the purified receptor was determined, and the specific activity of the purified preparation was estimated to be 13,100 pmol/mg of protein on the basis of amino acid composition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified receptors with or without radioiodination revealed a single, major band with an apparent Mr of 70,000 either by silver staining or radioautogram. The major band corresponded to the band which specifically bound [3H]propylbenzylcholine mustard (irreversible muscarinic ligand). The purified receptor showed essentially the same specificity for muscarinic ligands as unpurified receptors.     

Purification and Characterization of a Cryoprotective Protein (Cryoprotectin) from the Leaves of Cold-Acclimated Cabbage

We have purified a protein (cryoprotectin) from the leaves of cold-acclimated cabbage (Brassica oleracea L.) that protects thylakoids from nonacclimated spinach (Spinacia oleracea L.) against freeze-thaw damage. The procedure involves precipitations by heat, ammonium sulfate, and the glycosaminoglycan heparin and column chromatography on Polyamide 6 and a C18 reverse-phase matrix. After reverse-phase chromatography we obtained a single band of an apparent molecular mass of 7 kD when fractions that showed cryoprotective activity were analyzed by sodium dodecyl sulfate gel electrophoresis and silver staining. Gel-filtration experiments confirmed that the active protein is a monomer of 7 kD native molecular mass. This 7-kD protein could be purified only from cold-acclimated cabbage, but not from plants grown under nonacclimating conditions. Using peroxidase-labeled lectins, we show that cryoprotectin is a glycoprotein and that the saccharide moiety contains [alpha]1-3-linked fucose.     

Purification of L-glutamate decarboxylase by affinity chromatography

L-Glutamate decarboxylase (L-glutamate 1-carboxy-lyase, EC 4.1.1.15) from rat brain synaptosomal extract was partially purified by affinity chromatography. On further purification by DEAE-Sephadex A 50 and Sephadex G-200, L-glutamate decarboxylase was purified to greater extent. It was found that a single affinity chromatography by appropriate elution gave a highly purified protein giving a single band of high specific activity on polyacrylamide gradient gel slab electrophoresis with minimal contamination. Substrate specificity of the purified enzyme was modified in the presence of 6-azauracil or phenylalanine resulting in decreased specificity to L-glutamate and increased specificity to L-aspartate.     

Purification and identification of the functional sodium- and chloride-coupled gamma-aminobutyric acid transport glycoprotein from rat brain

Using the reconstitution conditions developed recently (Radian, R., and Kanner, B. I. (1985) J. Biol. Chem. 260, 11859-11865) we have now purified the sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain to apparent homogeneity. A partially purified transporter preparation was passed over wheat germ agglutinin-Sepharose 6MB and non-bound proteins were washed away. The transport activity, as expressed upon reconstitution of the protein into liposomes, was eluted by a solution containing Triton X-100 and N-acetylglucosamine. The specific transport activity was increased almost 400-fold over that of the crude extract. Taking into account an approximately 2.5-fold inactivation during the lectin column chromatography, the actual purification is about 1000-fold. Sodium dodecyl sulfate-polyacrylamide electrophoresis of the active fractions revealed one band of 80 kDa and small amounts of a band which ran at an apparent molecular mass of 160 kDa. The ratio between the two could be experimentally changed such as, for instance, by lyophilization. Polyclonal antibodies were prepared against the 80-kDa band which also cross-reacted with the 160-kDa band, indicating that the latter apparently represents a dimer form of the first. Using Protein A-Sepharose Cl-4B and the antibody against the 80-kDa band, we were able to quantitatively immunoprecipitate the potential gamma-aminobutyric acid transport activity from a crude transporter preparation. The pure transporter preparation exhibited the same features of the transporter in synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogeneity, affinity, and efflux and exchange properties. We conclude that the 80-kDa band represents the gamma-aminobutyric acid transporter.     

Characterization of a Mg2+-dependent phosphatase from turkey gizzard smooth muscle

A Mg2+-dependent phosphatase has been purified to apparent homogeneity from turkey gizzard smooth muscle. The enzyme has a Mr = 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 44,500 as determined by sedimentation equilibrium centrifugation under nondenaturing conditions. Using polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate all of the phosphatase activity was found to migrate as a single band, subsequently shown to have an Mr = 43,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is inactive in the absence of Mg2+ and maximum activity is reached at a free concentration of 12 mM Mg2+. Mn2+ can replace Mg2+, but the activity is only about one-fifth of that found with 12 mM Mg2+. NaF and the nucleotides ATP, ADP, and AMP inhibit phosphatase activity. This inhibition appears to be independent of their ability to bind Mg2+. The phosphatase purified from turkey smooth muscle appears to be identical with that purified from canine heart (Binstock, J. F., and Li, H. C. (1979) Biochem. Biophys. Res. Commun. 87, 1226-1234) and rat liver (Hiraga, A., Kikuchi, K., Tamura, S., and Tsuiki, S. (1981) Eur. J. Biochem. 119, 503-510).     

Cross-linking of iodine-125-labeled, calcium-dependent regulatory protein to the Ca2+-sensitive phosphodiesterase purified from bovine heart.

The calcium-dependent regulatory protein (CDR).Ca2+ sensitive cyclic nucleotide phosphodiesterase was purified to apparent homogeneity from bovine heart by using ammonium sulfate fractionation, DEAE-ceelulose chromatography, and CDR-Sepharose affinity chromatography. The enzyme was purifed 13 750-fold with a 10% yield and a specific activity of 275 mumol of cAMP min-1 mg-1. The purified enzyme ran as a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 57 000. Phosphodiesterase activity was stimulated 10-fold by Ca2+ and CDR with half-maximal activation occurring at 9 ng/assay. [125I]CDR was cross-linked to the purified phosphodiesterase by using dimethyl suberimidate Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cross-linked products revealed a number of discrete 125I-labeled bands. The molecular weights of the cross-linked products indicate that the stoichiometry of the phosphodiesterase complex is A2C2, where A is the phosphodiesterase catalytic subunit and C is the calcium-dependent regulatory protein.     

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.