Purification and characterization of a human kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide and biologically active peptides

An enzyme hydrolyzing succinyl trialanine-4-nitroanilide was extracted from human kidney homogenate and purified by means of gel filtration on Sepharose CL-4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme consists of a single peptide, and its molecular weight was estimated to be about 125 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme cleaved the substrate at the bond between succinyl dialanine and alanine-4-nitroanilide and showed a Km value of 2.1 mM at the optimal pH of 8.0. The activity was increased by Ca2+ and Mg2+, but was inhibited by phosphoramidon and ethylenediaminetetraacetic acid. The enzyme cleaved the oxydized insulin B chain, angiotensinogen tetradecapeptide, angiotensin I, angiotensin II, angiotensin III, [Sar1,Ala8]-angiotensin II, bradykinin, des-Pro2-bradykinin, Leu5-enkephalin, Met 5-enkephalin, [D-Ala2,Met5]-enkephalinamide and [D-Ala2-Met5]-enkephalin, but did not cleave [D-Ala2,D-Leu5]-enkephalin. The bonds on the amino side of the hydrophobic amino acids of the peptides were cleaved by the enzyme.     

Proteolysis in eukaryotic cells. Proteinase yscE, a new yeast peptidase

A new peptidase, which we call proteinase yscE, was purified from the yeast Saccharomyces cerevisiae. The enzyme cleaves the synthetic substrates Cbz-Gly-Gly-Leu-4-nitroanilide, Cbz-Ala-Ala-Leu-4-nitroanilide, and Suc-Phe-Leu-Phe-4-nitroanilide (Cbz and Suc are defined as benzyloxycarbonyl and succinyl, respectively) at the 4-nitroanilide bond and exhibits a slight activity against [3H]methylcasein. Optimum pH for cleavage of the chromogenic substrates is found to be in the range of 8.2 to 8.6. The purified enzyme has an apparent Stokes radius of Rs = 75.2 A as judged by gel chromatography and is composed of subunits. Mercurials were found to be strong inhibitors of the enzyme activity.     

Proteinase yscD. Purification and characterization of a new yeast peptidase

A newly recognized peptidase, designated proteinase yscD, was purified from the yeast Saccharomyces cerevisiae. The enzyme cleaves the Pro-Phe bond of the synthetic peptide substrate Bz-Pro-Phe-Arg-4-nitroanilide and the Ala-Ala bond of Ac-Ala-Ala-Pro-Ala-4-nitroanilide, Ac-Ala-Ala-Pro-Phe-4-nitroanilide, and MeO-Suc-Ala-Ala-Pro-Met-4-nitroanilide with high efficiency (Bz-, Ac-, and MeO-Suc are defined as benzoyl, acetyl, and methoxy-succinyl, respectively). [3H]Methylcasein does not serve as a substrate. Optimum pH for cleavage of Bz-Pro-Phe-Arg-4-nitroanilide is in the range of 6.5 to 7; for Ac-Ala-Ala-Pro-Ala-4-nitroanilide the range is between 5.75 and 6. For MeO-Suc-Ala-Ala-Pro-Met-4-nitroanilide the pH optimum was found to be 5.5. The purified enzyme has an apparent Stokes radius of Rs = 37.9 A as judged by gel chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates a molecular weight of approximately 83,000 for the enzyme. Mercurials and EDTA were found to be potent inhibitors of proteinase yscD activity.     

Enkephalinase from rat kidney. Purification, characterization, and study of substrate specificity

"Enkephalinase," a membrane-bound peptidase hydrolyzing the Gly3-Phe4 amide bond of enkephalins, initially characterized in brain, was purified from a rat kidney microsomal fraction. After differential solubilization with Triton X-100, the use of DEAE-Sephadex, concanavalin A, and hydroxylapatite chromatography led to a 2000-fold purification, close to homogeneity. Renal enkephalinase appears to be a glycoprotein Mr = 92,000-95,000 with catalytic properties and sensitivity to chelating agents and inhibitors (Thiorphan, phosphoramidon) very similar to those of the cerebral enzyme. The enzyme co-purified until the final step with "renal brush-border neutral proteinase" (EC 3.4.24.11) activity assayed with 125I-insulin B chain as substrate and displaying similar sensitivity to inhibitors. The specificity of the purified enkephalinase has been studied using either peptides derived from the enkephalins or model peptides of general formula (Ala)m-Tyr-(Ala)n as substrates. In all cases the bond cleaved was that involving the amino group of an aromatic residue, specificity being also defined by the nature of the neighboring residue on the COOH-terminal side. A free carboxyl in the latter residue was essential in the two series of substrates, indicating that enkephalinase more efficiently functions as a dipeptidyl carboxypeptidase than as an endopeptidase.     

A membrane-bound metallo-endopeptidase from rat kidney hydrolyzing parathyroid hormone. Purification and characterization

A metallo-endopeptidase, which appears to be an integral membrane protein of rat kidney, was purified to homogeneity by a series of standard chromatographic procedures. This enzyme significantly hydrolyzed human parathyroid hormone [hPTH(1-84)] and a synthetic substrate Suc-Leu-Leu-Val-Tyr-Mec (Suc = succinyl, Mec = 4-methyl-coumarinyl-7-amide). The purified enzyme had apparent molecular masses of 250 kDa on gel filtration, and 88 kDa and 245 kDa on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions, respectively. Its pH optimum for activity was 8.0-8.5 and its isoelectric point was pH 4.9. Its activity was inhibited by EDTA, EGTA and o-phenanthroline, but not by phosphoramidon. The metal-depleted enzyme was reactivated by the addition of metal ions. The enzyme was also inhibited by chymostatin and eglin C, and by thiol compounds. Of the synthetic substrates examined, the enzyme hydrolyzed only Suc-Leu-Leu-Val-Tyr-Mec, one of the synthetic substrates for alpha-chymotrypsin. It did not hydrolyze synthetic substrates with less than four amino acid residues with tyrosine in the P1 position. The enzyme hydrolyzed hPTH and reduced hen egg lysozyme but did not hydrolyze azocasein or [3H]methyl-casein. NH2-terminal amino acid sequence analyses of the degradation products of hPTH(1-84) and reduced hen egg lysozyme by the purified enzyme revealed that the enzyme preferentially cleaved these peptides at peptide bonds flanked by hydrophilic amino acid residues. Amino acid analyses showed that the main degradation products of PTH were hPTH(17-29), hPTH(30-38) and hPTH(74-84). The ability of the enzyme to hydrolyze peptide bonds flanked by hydrophilic amino acid residues and its inability to degrade azocasein distinguish it from several other kidney endopeptidases reported, such as endopeptidase 24.11 and meprin.     

Purification and Partial Characterization of an Aminopeptidase from Lactobacillus lactis

A surface-bound aminopeptidase of Lactobacillus lactis cells was solubilized with lysozyme, and the extract was subjected to streptomycin sulfate precipitation, ammonium sulfate fractionation, chromatography on Sephadex G-100 and diethylaminoethyl-Sephadex A-50, and preparative polyacrylamide gel electrophoresis. The purified enzyme was homogeneous in disc electrophoretic analysis and consisted of a single polypeptide chain with a molecular weight of 78,000 to 81,000. The optimal pH and optimal temperature for enzyme activity were 6.2 to 7.2 and 47.5 degrees C, respectively, for l-lysine-4-nitroanilide as the substrate. The enzyme was activated by Co and Zn ions and inhibited by Cu, Hg, and Fe ions and by the metal-complexing reagents ethylenediaminetetraacetic acid, 1,10-phenanthroline, and alpha,alpha'-dipyridyl. Higher concentrations of substrate and hydrolysis products also inhibited the activity of the enzyme. The aminopeptidase had broad substrate specificity and hydrolyzed many amino acid arylamides and many peptides with unsubstituted NH(2)-terminal amino acids.     

Sex-related difference in vitamin D3 25-hydroxylase of rat liver microsomes

Cholecalciferol 25-hydroxylase was partially purified by polyethylene glycol fractionation and chromatographies on octylamino-Sepharose and hydroxylapatite columns starting from the liver microsomes of female rats, and compared with P-450cc25 purified from the liver microsomes of male rats (Hayashi, et al. (1986) J. Biochem. 99, 1753-1763). On octylamino-Sepharose 4B column chromatography, most of the activity was recovered in the fraction eluted with 0.08% Emulgen 913 in the case of the male enzyme, whereas the female enzyme was recovered in the fraction eluted with 0.2% Emulgen. Anti-cc25 antibodies against purified male P-450cc25 inhibited the 25-hydroxylation activity of male polyethylene glycol (PEG) fraction and partially purified male enzyme, but did not inhibit the activities of the corresponding female fractions. The antibodies formed a single precipitation line with male P-450cc25, but did not form a precipitation line with partially purified female 25-hydroxylase on immuno-diffusion. These observations indicated that the vitamin D3 25-hydroxylase in female rat liver microsomes is a different entity from that of male rats.     

Purification of a membrane-bound neutral endopeptidase from rat kidney and its activation by polyamines

An endopeptidase which cleaves succinyl trialanine p-nitroanilide (Suc(Ala)3-pNA) into succinyl dialanine and alanine p-nitroanilide (Ala-pNA) was solubilized from a microsomal membrane fraction of rat kidney with Nonidet P-40 following treatment with 1 M KCl and Brij 35. The solubilized enzyme was purified to homogeneity by DEAE-Sephadex chromatography, Sepharose CL-6B gel filtration and sucrose gradient centrifugation. The final enzyme preparation had a specific activity of 1.69 mumol/min/mg protein, representing about 140-fold purification over the starting membrane. The enzyme hydrolyzes Suc(Ala)3-pNA with a Km value of 0.28 mM and a Vmax value of 1.3 mumol/min. The molecular weight of the undenatured enzyme was estimated to be 360,000 by gel filtration on a Sepharose CL-6B column and that of the denatured enzyme to be 92,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, revealing the presence of a single polypeptide chain. The enzyme was markedly activated by polyamines, producing increases in the values of both Km and Vmax. Comparatively less activation was found in the presence of some monovalent cations and Ca2+. The activation by polyamines was inversely proportional to the concentration of monovalent cations, but Ca2+ and polyamines seemed to stimulate additively.     

Isolation, characterization, and localization of antigen gamma, a serine proteinase of the "kallikrein-family" in the rat submandibular gland

A trypsin-like serine proteinase, antigen gamma, immunologically partially identical to glandular kallikrein when run against anti-rat glandular kallikrein antiserum in immunoelectrophoresis, was purified from the rat submandibular gland. The enzyme was purified by a two-step chromatography procedure, ionexchange chromatography followed by gel filtration. The criteria for purity were one band in SDS-polyacrylamide gel electrophoresis and in immunoelectrophoresis, respectively. Antigen gamma had a molecular mass of 25,000 Da and consisted of two polypeptide chains with molecular masses of 14,000 and 11,000 Da. The preparation contained several isoenzymes with pI ranging from 4.1 to 4.5. The enzyme showed high specific enzyme activity against the substrate D-valyl-L-leucyl-L-arginine-4-nitroanilide (S-2266), some trypsin-like and kininogenase activity, but no angiotensin converting enzyme, kininase, or tonin activity. Amidolytic activity was increased and stabilized by the presence of detergent in the assay buffer. The pH-optimum of antigen gamma amidolytic activity was about 10. Antigen gamma was inhibited by SBTI and PMSF, whereas aprotinin had to be added in a more than 100 times higher concentration than for glandular kallikrein. The binding pattern of antigen gamma to plasma proteins was different from that of tonin and glandular kallikrein. Antiserum against antigen gamma was raised in rabbits and characterized against rat submandibular gland homogenate. Immunohistochemistry showed antigen gamma in the secretory granules of the submandibular gland granular tubular cells but only adhering to the luminal cell wall in the striated and main excretory ducts. Antigen gamma was not detected in the sublingual or parotid gland or in the kidney. Antigen gamma was demonstrated by immunoelectrophoresis in rat submandibular gland saliva. The concentration was higher in sympathetically than in parasympathetically induced secretion.     

Leukotriene A4 hydrolase: an anion activated peptidase.

The peptidase activity of leukotriene A4 hydrolase purified from human leukocytes has been characterized, utilizing synthetic amides as substrates. The enzyme was stimulated by several monovalent anions. Thiocyanate ions were most effective followed by chloride and bromide ions. In phosphate buffer alone the peptidase activity towards alanine-4-nitroanilide was barely detectable and addition of 100 mM NaCl increased the specific activity more than 20-fold. Increasing the concentration of NaCl (or NaSCN) did not significantly affect the apparent Km for the substrate alanine-4-nitroanilide, but resulted in a dose dependent increase of Vmax. The stimulatory effect of these anions on the reaction velocities appeared to obey saturation kinetics and thus indicated the presence of an anion binding site. Apparent affinity constants for chloride and thiocyanate ions were calculated to 100 and 50 mM, respectively. In contrast to the effect on the peptidase activity, no chloride-stimulation could be detected of the epoxide hydrolase activity of this enzyme, i.e., the conversion of leukotriene A4 into leukotriene B4. In conclusion, the results indicate that under physiological conditions, chloride ions may selectively stimulate the peptidase activity of LTA4 hydrolase. Also, the differences in chloride concentrations between cellular compartments suggest that a possible proteolytic function of the enzyme may be limited to the extracellular space.     

Hydrolysis of substance P and its analogs by angiotensin-converting enzyme from rat lung. Characterization of endopeptidase activity of the enzyme

Hydrolysis of substance P and nine kinds of substance P analogs by angiotensin-converting enzyme highly purified from rat lung was examined by using amino-group fluorometry and high-performance liquid chromatography. The enzyme hydrolyzed substance P and several analogs, notwithstanding that they did not contain free C-terminal residues. The analyses of cleavage products separated by high-performance liquid chromatography indicated that the enzyme hydrolyzed substance P and its analogs mainly at the bond between Phe8-Gly9 and also at another bond, possibly between Gly9-Leu10, to a lesser extent by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action. The analogs that had D-amino acid residues substituted at the presumed cleavage sites were scarcely hydrolyzed. It was further found that (Pyr6)-fragment (6-11) was hydrolyzed by the enzyme more efficiently than the other fragment-type analogs and was cleaved at a single bond by the endopeptidase activity of the enzyme. Therefore, this fragment was used as a substrate in order to characterized the endopeptidase activity of the enzyme by employing fluorometry. The activity was dependent on chloride ion, and was inhibited by captopril, MK-421, and EDTA. Thus, the endopeptidase activity of the enzyme showed properties similar to those of the dipeptidyl carboxypeptidase activity of the enzyme.     

Partial purification of γ-glutamyltransferase from human brain microvessels

Two forms of gamma-glutamyltransferase from human brain cortex microvessels were partially purified by gel permeation and ion-exchange and group-affinity chromatography. The specific activity of the purified preparations was 320-fold (detergent form) and 830-fold (proteolytic form) higher than that of the enzyme in the brain cortex homogenate. The relative molecular mass of the proteolytic form of the enzyme was about 90,000 as determined by gel permeation chromatography. The major part of the enzyme (about 80%) was absorbed on Con A-Sepharose 4B. The pH optima for transfer reactions with -glutamyl-4-nitroanilide as donor and glycylglycine andl-cystine as acceptors were in the range of 8.2 to 9.0. The studied enzyme was inhibited by a mixture ofl-serine and borate and by bromcresol green.     

Comparison of the properties of purified mitochondrial and cytosolic rat kidney transamidinase

1. Mitochondrial rat kidney transamidinase was solubilized by two extractions with the surfactant Zwittergent 3-14. 2. Mitochondrial and cytosolic forms of rat kidney transamidinase were purified by chromatography on DEAE-Trisacryl M, phenyl-Sepharose Cl-4B and hydroxylapatite columns. 3. The specific activity of purified mitochondrial enzyme was significantly higher than purified cytosolic enzyme. 4. The subunit molecular mass, the electrophoretic mobility under nondenaturing conditions, and the activation energy were similar for purified mitochondrial and cytosolic transamidinase.     

Purification and some properties of camel carboxypeptidase B

A carboxypeptidase B-like enzyme was purified 116-fold with a recovery of activity of 29% from a crude extract of camel pancreas by a four-step procedure consisting of two anion exchange chromatographies in succession, gel filtration and hydrophobic interaction chromatography. The enzyme was homogeneous on SDS and non-denaturing gel electrophoresis and on gel isoelectric focusing. Its molecular mass was found to be 31.5 kDa and its isoelectric point was estimated as 6.1. It was active towards a number of substrates that are cleaved by carboxypeptidases B from other species and was also susceptible to inhibition by inhibitors of such enzymes. The camel enzyme showed a pH optimum of 8.0 and it was seen to be a relatively potent kinase in vitro. The enzyme purified in this study was very similar to carboxypeptidases B isolated from other species in size, charge, substrate specificity and susceptibility to inhibition and thus it can be identified as camel carboxypeptidase B.     

Purification and characterization of proteinase yscJ, a new yeast peptidase.

A newly recognized peptidase, designated proteinase yscJ, was purified from the yeast Saccharomyces cerevisiae. The enzyme is of non-vacuolar origin and cleaves the Tyr-Lys bond of the synthetic peptide substrate Cbz-Tyr-Lys-Arg-NH-Ph (Cbz, benzyloxycarbonyl; NH-Ph, 4-nitroanilide) and the Glu-Lys bond of the substrate Boc-Glu-Lys-Lys-NH-Mec (Boc, butoxycarbonyl; Mec, 4-methylcoumarinyl) with high efficiency. Optimum pH for cleavage of Cbz-Tyr-Lys-Arg-NH-Ph is in the range 7.0-7.5. The purified enzyme has a molecular mass of approximately 58 kDa, as judged by gel filtration on a Superose 12 FPLC column. Mercury compounds and EDTA were found to be potent inhibitors of proteinase yscJ activity.     

Purification and properties of an extracellular leucine aminopeptidase from the Bacillus sp. N2

A halophilic bacterium was isolated from fermented anchovy sauce and identified as Bacillus species. An extracellular leucine aminopeptidase from Bacillus sp. N2 was purified to homogeneity using four successive purification steps. The enzyme has a native molecular mass of about 57 000 Da using FPLC gel filtration analysis and a molecular mass of 58 000 Da using SDS-polyacrylamide gel electrophoresis. This monomeric leucine aminopeptidase showed maximum enzyme activity at pH 9·5. The optimum temperature was 50 °C when L -Leu- p -nitroanilide was the substrate. The leucine aminopeptidase was inactivated by 1,10-phenanthroline, dithiothreitol and sodium dodecyl sulphate. Enzyme activity was increased by addition of Co²⁺. It is likely that Co²⁺ plays an important role in the catalysis or stability of the Bacillus sp. N2 leucine aminopeptidase. Sodium chloride (0–4·5 mol l⁻¹) increased the hydrolytic activity towards L -Leu- p -nitroanilide. The N-terminal amino acid sequence was Glu-Arg-Glu-Leu-Pro-Phe-Lys-Ala-Lys-His-Ala-Tyr-Ser-Thr-Ile. The purified enzyme had a high specificity for L -Leu- p -nitroanilide.     

Difference in renal alpha-galactosidase levels in male and female Chinese hamsters (Cricetulus griseus).

1. The activity of alpha-galactosidase was found to be significantly higher in the kidney of female than that of male Chinese hamsters in a highly inbred colony but its activity in liver, heart and spleen remained similar between female and male animals. 2. Partially purified renal alpha-galactosidase by sequential column chromatography on Sepharose 6B and DEAE-Sepharose CL-6B showed identical elution profiles, pH optima (4.5), KmS (4.4 mM) and heat-inactivation curves between enzymes of male and female animals. 3. Thus, the observed higher activity of renal alpha-galactosidase in the females was due to elevated enzyme concentration, not a result of enzyme polymorphism.     

Effect of hydroxynitrobenzylation of tryptophan-177 on reactivity of active site cysteine-25 in papain

It is known that the enzymatic activity of papain (EC 3.4.22.2) toward α-N-benzoyl-l-arginine p-nitroanilide can be substantially increased by hydroxynitrobenzylation of Trp-177 through reaction of the enzyme with the active site-directed reagent, 2-chloromethyl-4-nitrophenyl (N-carbobenzoxy)glycinate (S.-M. T. Chang and H. R. Horton, 1979, Biochemistry18, 1559–1563). To determine the effect of such hydroxynitrobenzylation on the nucleophilicity of the essential thiol group at the active site of the enzyme, rates of inactivation by S_N2 reactions of Cys-25 with chloroacetamide and chloroacetate and by Michael addition of Cys-25 to N-ethylmaleimide were monitored. The kinetics revealed that, at pH 6.5, the reactivities of the sulfhydryl group of hydroxynitrobenzylated papain with chloroacetamide and with N-ethylmaleimide are 24 and 27% greater than those of the sulfhydryl group of native papain. At pH 7.1, the rate enhancements are 34 and 39%, respectively. These increases in reactivity of Cys-25 as an attacking nucleophile appear to account for the increased catalytic activity of hydroxnitrobenzyl-papain toward an oligopeptide substrate, α-N-benzoyl-l-phenylalanyl-l-valyl-l-arginine p-nitroanilide, and toward an ester substrate, N-carbobenzoxyglycine p-nitrophenyl ester. However, the presence of the hydroxynitrobenzyl reporter group provides substantially greater improvement (250%) in enzymatic efficiency toward α-N-benzoyl-l-arginine p-nitroanilide, apparently by blocking nonproductive binding of this substrate to the enzyme. Fluorescence changes accompanying the various chemical modifications are interpreted in terms of a charge-transfer interaction between the imidazolium ion of His-159 and the indole moiety of Trp-177 in the active form of native papain, which should help to stabilize the catalytically essential mercaptide-imidazolium ion-pair (Cys-25, His-159).     

Purification and Characterization of an Extracellular Alkaline Serine Protease from Aspergillus terreus (IJIRA 6.2)

An extracellular alkaline serine protease has been purified from Aspergillus terreus (IJIRA 6.2). The purification procedure involved chromatography on DEAE-Sephadex A25, phosphocellulose, hydroxyapatite, casein-Sepharose, gel filtration on Sephacryl-S-300 and by glycerol density gradient centrifugation. The enzyme was further purified to apparent homogeneity through a combination of electrophoresis in polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) with or without protease substrate (gelatin) and subsequent regeneration of its activity in situ by removal of SDS. The active enzyme was visualized in a zymogram or on the basis of protease activity exhibited on an X-ray film. The protein in the unstained segment of the gel was electroeluted. The eluted protein with protease activity exhibited a molecular mass of 37,000-daltons on electrophoresis in SDS-polyacrylamide gel. A sedimentation coefficient of 3.2S was obtained by glycerol density gradient contrifugation. Maximum activity of protease was observed at pH 8.5 and at 37°C. Purified protease was active between pH 5.5 and 9.5 and was found to be stable up to 60°C. With Na-caseinate, the K _m of the purified protease was found to be 0.055 mM. Antipain, phenylmethane sulfonyl fluoride, and chymostatin served as non-competitive inhibitors. Substrate specificity was determined by using a synthetic chromogenic peptide containing N-P-Tosyl-Gly-Pro-Arg-p-nitroanilide. Results showed that the protease cleaved the peptide on the -COOH end of arginine residue. Received: 8 October 1999 / Accepted: 3 November 1999     

Rat epididymal alpha-D-mannosidase: purification, carbohydrate composition, substrate specificity, and antibody production

Two alpha-D-mannosidases have previously been identified in rat epididymis. This communication reports the purification and characterization of the "acid" alpha-D-mannosidase. The enzyme was purified over 1000-fold to near homogeneity by acetone and (NH4)2SO4 precipitation followed by ion-exchange and hydroxylapatite chromatography. The molecular weight of the enzyme was estimated to be 220,000 by gel filtration. Polyacrylamide gel electrophoresis of the native enzyme under two conditions of buffer and pH showed a single band when stained for protein while electrophoresis under denaturing conditions resulted in bands of apparent Mr 60,000 and 31,000. The enzyme is a glycoprotein containing about 5.6% hexose. In addition to mannose (3.1%) and glucosamine (2.0%), the enzyme also contained small amounts of glucose, fucose, and galactose. Chemical analysis indicated the absence of sialic acid. The substrate specificity of the purified enzyme was investigated using linear and branched mannose-containing oligosaccharides. The enzyme cleaved linear oligosaccharides [Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc and Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc] very efficiently. However, little or no activity was observed toward high mannose oligosaccharides (Man9GlcNAc through Man5GlcNAc) or the branched trimannosyl derivative Man3GlcNAc. This specificity is very similar to that observed with rat kidney lysosomal alpha-D-mannosidase. Additional evidence that the epididymal enzyme is essentially a lysosomal alpha-D-mannosidase is the fact that polyclonal antibody prepared against the purified epididymal enzyme cross-reacted with lysosomal alpha-D-mannosidase from several rat tissues and with acidic alpha-D-mannosidase of a human cell line, results suggesting that the antibody will be useful in studying the biosynthesis and turnover of lysosomal alpha-D-mannosidases in at least two species.