Acid phosphatase in rat liver lysosomal membranes: purification and characterization

Acid phosphatase associated with rat liver lysosomal membranes (M-APase) was purified about 4,200-fold over the homogenate with 10% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included; preparation of lysosomal membranes, solubilization of the membranes with 1% Triton X-100, immunoaffinity chromatography, and gel filtration with FPLC equipped with a Sephacryl S-300HR column. The molecular weight, estimated by gel filtration through TSK SW 3000G, was approximately 320K and SDS gel electrophoresis showed that the enzyme is composed of four identical subunits with an apparent molecular weight of 67K. The enzyme contains about 24.3% carbohydrate consisting of mannose, galactose, fucose, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid in a molar ratio of 38:20:5:36:4:11, respectively. In addition, three soluble forms of acid phosphatase (C-APase I, II, and III) in lysosomal contents were separated from rat liver lysosomal contents with DEAE-Sephacel. These three enzymes were also purified using immunoaffinity chromatography followed by gel filtration. C-APase I, II, III, and M-APase have isoelectric points of 7.7-8.2, 6.6-7.0, 5.7-6.7, and 3.4-3.8, respectively. All four APases are sensitive to endo-beta-N-acetylglucosaminidase H. However, only C-APase III and M-APase are digestible with neuraminidase. Susceptibility of M-APase to neuraminidase in intact tritosomes was examined to study the topography of M-APase in tritosomal membranes. Neuraminidase susceptibility of M-APase was not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of dipeptidyl peptidase IV in rat liver lysosomal membranes.

Dipeptidyl peptidase IV (m-DPP IV) in rat liver lysosomal membranes was purified about 50-fold over the lysosomal membranes with 38% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The enzyme amounts to about 3% of lysosomal membrane protein constituents. The purification procedures included: extraction of lysosomal membranes by Triton X-100, WGA-Sepharose affinity chromatography, hydroxylapatite chromatography, ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. The enzyme (M(r) 240,000) is composed of two identical subunits with an apparent molecular weight of 110,000. The enzyme contains about 12.4% carbohydrate and the carbohydrate moiety was composed of mannose, galactose, fucose, N-acetylglucosamine, and neuraminic acid in a molar ratio of 14:17:2:24:11. Susceptibility to neuraminidase and immunoreactivity of the enzyme in intact tritosomes were examined to study the topology of the enzyme in tritosomal membranes. Neuraminidase susceptibility and immunoreactivity of the enzyme were not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock. This result indicated that both the oligosaccharide chains and the main protein portion of the enzyme are on the inside surface of the tritosomal membranes. Subcellular localization of DPP IV was determined by means of enzyme immunoassay, which indicated that bile canalicular membranes and lysosomal membranes are the major sites of localization, and DPP IV activity in lysosomes was separated into a membrane bound form (60%) and a soluble form (40%). Immunoelectron microscopy clearly confirmed that DPP IV occurs not only in the bile canalicular domain but also in the lysosomes of rat liver.     

Mechanisms of a conversion from membrane associated lysosomal acid phosphatase to content forms

We reported that membrane-associated APase (M-APase) is anchored in the lipid bilayer through its hydrophobic sequence close to the COOH-terminus [Biochem. Biophys. Res. Commun. (1989) 162, 1044-1053] and is released from lysosomal membranes into the lysosomal contents by limited proteolysis with cathepsin D [J. Biochem. (1990) 108, 287-291]. We here report the conversion process of M-APase to three forms of the content enzyme (C-APase I, II, and III) by assigning the COOH-terminus of each APase in lysosomes. The purified M-APase (67 kDa) was subjected to COOH-terminal determination after digestion with cathepsin D. The COOH-terminus of cathepsin D-digested M-APase (65 kDa) ended at the position of the 382nd leucine residue. The COOH-termini of C-APase I (48 kDa) and III (64 kDa) were also determined. Since the two enzymes ended at the same position of the 373rd alanine residue, this COOH-terminal is 9 amino acid residues shorter than that of cathepsin D-digested M-APase. Then, we compared NH2-terminal sequences of the three enzymes, and found that those of three enzymes are exactly the same. Therefore, protein portions of C-APase I and III proved to be identical. The above results indicate that in lysosomes M-APase is first hydrolyzed between amino acid residues 382 and 383 by cathepsin D, and after solubilization, the enzyme is converted to C-APase III by losing 9 amino acid residues by lysosomal carboxypeptidase(s). Molecular weight differences among three C-APases (III, 64 kDa; II, 55 kDa; I, 48 kDa) probably are due to different degrees of carbohydrate chain degradations as reported previously [J. Biochem. (1989) 105, 449-456].     

Purification and characterization of an 85 kDa sialoglycoprotein in rat liver lysosomal membranes.

Sialoglycoprotein with a molecular mass of 85 kDa (LGP85) was purified from rat liver lysosomal membranes with a 0.9% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The purification procedures included: preparation of lysosomal membranes, elimination of LGP107 and LGP96 with immunoaffinity columns, WGA-Sepharose affinity chromatography, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. LGP85 contains about 22.8% carbohydrate and the carbohydrate moiety is composed of mannose, galactose, fucose, glucosamine, galactosamine, and neuraminic acid, in a molar ratio of 40:20:2:23:3:13. Susceptibility to neuraminidase and immunoreactivity of the protein in intact tritosomes were examined to study the topology of the protein in tritosomal membranes. Neuraminidase susceptibility and immunoreactivity of the protein were not observed in intact tritosomes until the tritosomes had been disrupted by osmotic shock. These observations suggest that both oligosaccharide chains and the main protein portion of the protein are located on the interior surface of the tritosomal membranes. Subcellular localization of LGP85 was determined using enzyme immunoassay. The lysosomes seem to be the major location. LGP85 in the lysosomes was divided into the membrane bound form (90%) and the soluble form (10%). Immunoelectron microscopy clearly confirmed that the localization of LGP85 is mainly confined to lysosomes.     

Molecular properties of the Factor V-activating enzyme from Russell's viper venom.

The protease from Russell's viper venom that activates Factor V was purified by gel filtration on Sephadex G-150 and ion exchange column chromatography on sulfopropyl (SP)-Sephadex C-50. The purified enzyme is a glycoprotein containing 6% carbohydrate. It migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 29,000. A minimum molecular weight of 27,200 was determined by sedimentation equilibrium in the presence of 6 M guanidine hydrochloride. The enzyme is composed of a single polypeptide chain possessing an NH2-terminal sequence of Val-Val-Gly-Gly-Asp-Glu-Cys-Asn-Ile-Asn-Glu-His-Pro-Ile. The specific activity of the Factor V activator toward tosyl-L-arginine methyl ester and D-phenylalanyl-L-pipecolyl-L-arginyl-p-nitroanilide was 380 and 11 nmol/min/mg, respectively. The esterase and coagulant activities of the enzyme were readily inhibited by diisopropyl fluorophosphate. The enzyme was not inhibited by bovine antithrombin III in the presence or absence of heparin. The amino acid and carbohydrate compositions of the enzyme are also reported.     

Purification and characterization of (Ca2+-Mg2+)-ATPase in rat liver lysosomal membranes.

A (Ca(2+)-Mg2+)-ATPase associated with rat liver lysosomal membranes was purified about 300-fold over the lysosomal membranes with a 7% recovery as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included: preparation of lysosomal membranes, solubilization of the membrane with Triton X-100, WGA-Sepharose 6B, Con A-Sepharose, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass, estimated by gel filtration with Sephacryl S-300 HR, was approximately 340 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular mass of 85 kDa. The isoelectric point of the purified enzyme was 3.6. The enzyme had a pH optimum of 4.5, a Km value for ATP of 0.17 mM and a Vmax of 71.4 mumol/min/mg protein at 37 degrees C. This enzyme hydrolyzed nucleotide triphosphates and ADP but did not act on p-nitrophenyl phosphate and AMP. The effects of Ca2+ and Mg2+ on the ATPase were not additive, thereby indicating that both Ca2+ and Mg(2+)-ATPase activities are manifested by the same enzyme. The (Ca(2+)-Mg2+)-ATPase differed from H(+)-ATPase in lysosomal membranes, since the enzyme was not inhibited by N-ethylmaleimide but was inhibited by vanadate. The effects of some other metal ions and compounds on this enzyme were also investigated. The N-terminal 18 residues of (Ca(2+)-Mg2+)-ATPase were determined.     

Characterization of tyrosine hydroxylase from bovine adrenal medulla

Tyrosine hydroxylase purified to apparent homogeneity from the soluble fraction of bovine adrenal medulla had an apparent Mr of about 280,000 by Bio-Gel A-1.5m chromatography, and gave a single band with a Mr of 60,000 by sodium dodesyl sulfate polyacrylamide gel electrophoresis. The enzyme is considered to be composed of four identical subunits. Isoelectric point of purified enzyme was pH 6.0. The amino acid composition of the enzyme was characterized by fairly high contents of glutamic acid and alanine residues. The N-terminal amino acid was determined to be glutamic acid.     

Characterization of the purified multifunctional cellulase component of Penicillium funiculosum

Summary The endoglucanase component (CMCase I) ofPenicillium funiculosum cellulase was purified to apparent homogeneity by ultrafiltration and gel chromatography. It consists of a single polypeptide chain with a molecular weight of 56000 and is a glycoprotein. Viscometric and end-product analysis revealed the randomness of enzyme action. Multifunctional characteristic of CMCase I was studied with various carbohydrate substrates.NCL Communication No.: 4307     

Purification and characterization of the extracellular beta-glucosidase produced by Candida wickerhamii

Candida wickerhamii NRRL Y-2563 produced a cell-bound beta-glucosidase when grown in complex media containing 50 g of cellobiose per liter. The majority of the enzyme was located on the cell surface and was released into the supernatant upon treatment of intact cells with Zymolyase 60,000. Only about 10% of the total activity was associated with the cytoplasm. The enzyme was purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had an apparent native molecular mass of about 198,000 Da and appeared to be composed of two subunits with approximate molecular masses of 94,000 Da. The beta-glucosidase contained approximately 30.5% (w/w) carbohydrate. Mannose was the only detected neutral carbohydrate associated with the purified enzyme. The enzyme demonstrated optimal activity at a pH of 4.0 to 5.0. The Km of the purified beta-glucosidase was 6.74 X 10(-2) M for cellobiose and 4.17 X 10(-3) M for p-nitrophenyl-beta-D-glucopyranoside. Glucose did not appear to inhibit the enzyme.     

Purification and properties of a secreted and developmentally regulated alpha-L-fucosidase from Dictyostelium discoideum.

During its development the eukaryotic microorganisms Dictyostelium discoideum secretes an alpha-L-fucosidase (EC 3.2.1.51). In cells of the growth phase almost no alpha-L-fucosidase activity is detectable. The activity increases steadily up to the aggregation stage and accumulates also in the extracellular medium. The developmental regulation is mediated by pulsatile cAMP signals. The alpha-L-fucosidase was purified from extracellular medium. The isolation procedure started with concentration of the enzyme by batchwise anion-exchange chromatography and ammonium sulfate precipitation, followed by Sephacryl S-300 gel filtration and further purification by fast protein liquid chromatography on Mono Q, phenyl-Superose, and finally Superose 12. The purified preparation was found to be essentially free of activities of six other glycosidases also secreted by D. discoideum. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed one major band with an apparent molecular mass of 62 kilodalton. Gel filtration of the enzyme on a Superose 12 column was consistent with an active monomer. A monoclonal antibody was produced, which recognizes a carbohydrate epitope shared by all lysosomal enzymes in D. discoideum. The pH optimum of the alpha-L-fucosidase is at 3.7. The apparent Michaelis constant for p-nitrophenyl alpha-L-fucoside as substrate is 1.2 mM. The enzyme catalyzes preferentially the hydrolysis of alpha 1----6GlcNAc but also of alpha 1----2Gal and alpha 1----3Glc fucosyl linkages.     

Purification and properties of Neurospora crassa laccase

Extracellular Neurospora laccase (p-diphenol:oxygen oxidoreductase; EC 1.10.3.2) has been purified to apparent homogeneity by classical purification techniques. The enzyme, which consists of mainly one form, has a molecular weight of 64,800 and contains 11% carbohydrate. The ultraviolet, visible, and electron paramagnetic resonance spectra indicate that both type I and type II copper are present, as described for the Polyporus versicolor enzyme. With the exception of phloroglucinol, only para- and ortho-diphenols serve as effective substrates for the enzyme. Like the extracellular form, intracellular laccase is a glycoprotein as shown by its ability to bind to Concanavalin A Sepharose. Other studies, including gel filtration and ion-exchange chromatography, revealed no differences between the intracellular and extracellular enzymes, suggesting that intracellular laccase is destined for excretion by the cell.     

Characterization of the trimeric, self-recognizing Geodia cydonium lectin I

A D-galactose-specific lectin I was extracted from the sponge Geodia cydonium and purified by affinity chromatography. The molecular weight of lectin I as determined by high-pressure liquid gel chromatography, was found to be 36500 +/- 1300. Disc gel electrophoresis in the presence and in the absence of sodium dodecyl sulfate showed that lectin I is a trimer composed of three different subunits (Mr: 13800, 13000 and 12200); two of the three subunits are linked by one disulfide bond. Isoelectric focusing gave a pI of 5.6 for the native molecule and a pI of 4.4 and of 7.4 for the subunits. The three subunits carry carbohydrate side chains, composed of D-galactose (94%) and of arabinose (5%). Based on experiments with lectins, the terminal D-galactose residues are bound by beta 1 leads to 6 and/or beta 1 leads to 4 glycosidic linkages. The Geodia lectin I contains, besides two carbohydrate recognition sites, at least one receptor site for a second lectin I molecule.     

Use of immunoadsorbent column chromatography for improved purification of arylsulfatase B from human placenta.

A simple and rapid procedure involving immunoadsorbent column chromatography has been developed for the isolation of lysosomal arylsulfatase B from human placenta. Using this method, we purified the enzyme over 20,000-fold with better recovery (16%) compared to that achieved by the conventional procedure. The enzyme appeared to be homogeneous and had an apparent molecular weight of 58,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. The purified enzyme migrated as two bands with apparent molecular weights of 43,000 and 8,000 by reductive SDS-PAGE.     

A rat liver lysosomal membrane flavin-adenine dinucleotide phosphohydrolase: purification and characterization

An enzyme hydrolyzing flavin-adenine dinucleotide (FAD) to flavin mononucleotide and AMP was identified and purified from rat liver lysosomal (Tritosomal) membranes. The purified enzyme showed a single band on silver-stained denaturing gels with an apparent Mr 70,000. Periodate-Schiff staining after denaturing gel electrophoresis of whole membrane preparations revealed that this enzyme is one of the major glycoproteins in lysosomal membranes. FAD appeared to be the preferred substrate for the purified enzyme; equivalent concentrations of NAD or CoA were hydrolyzed at about one-half of the FAD rate. Negligible activity (less than or equal to 16%) was noted with ATP, TTP, ADP, AMP, FMN, pyrophosphate, or p-nitrophenylphosphate. The enzyme was inhibited by EDTA or dithiothreitol. It was stimulated by Zn, and was not affected by Ca or Mg ions, nor by p-chloromercuribenzoate. The pH optimum for FAD hydrolysis was 8.5-9 with an apparent Km of 0.125 mM. Antibodies prepared against the purified enzyme partially (50%) inhibited FAD phosphohydrolase activity in lysosomal membrane preparations but had no effect on the soluble lysosomal acid pyrophosphatase known to hydrolyze FAD. This enzyme could not be detected immunochemically in preparations of microsomes, Golgi, plasma membranes, mitochondrial membranes, or the soluble lysosomal fraction, suggesting that the enzyme is different from either soluble lysosomal acid pyrophosphatase or other FAD hydrolyzing activities in the liver cell.     

A Rapid Purification of L-Glutamic Acid Decarboxylase from the Brain of the Locust Schistocerca gregaria

L-Glutamic acid decarboxylase (GAD; EC 4.1.1.15) was purified to apparent homogeneity from the brain of the locust Schistocerca gregaria using a combination of chromatofocusing (Mono P) and gel filtration (Superose 12) media. The homogeneity of the enzyme preparation was established by native polyacrylamide gel electrophoresis (PAGE) with silver staining. The molecular weight of the purified enzyme was estimated from native gradient gel electrophoresis and gel filtration chromatography to be 97,000 +/- 4,000 and 93,000 +/- 5,000, respectively. When analysed by sodium dodecyl sulphate-PAGE, the enzyme was found to be composed of two distinct subunits of Mr 51,000 +/- 1,000 and 44,000 +/- 1,500. Tryptic peptide maps of iodinated preparations of these two subunits showed considerable homology, suggesting that the native enzyme is a dimer of closely related subunits. The purified enzyme had a pH optimum of 7.0-7.4 in 100 mM potassium phosphate buffer and an apparent Km for glutamate of 5.0 mM. The enzyme was strongly inhibited by the carbonyl-trapping reagent aminooxyacetic acid with an I50 value of 0.2 microM.     

Membrane-associated carbonic anhydrase purified from bovine lung

We found carbonic anhydrase activity associated with particulate fractions of homogenates of rat, rabbit, human, and bovine lungs. These membrane-associated carbonic anhydrases were remarkably stable in solutions containing sodium dodecyl sulfate (SDS). The bovine enzyme was dissolved with SDS and purified by affinity chromatography and gel filtration. The purified enzyme contains glucosamine, galactose, and sialic acid; it is at least 20% carbohydrate. The apparent molecular weight by SDS-polyacrylamide gel electrophoresis (52,000) may be higher than the actual molecular weight due to the presence of carbohydrate. The enzyme contains cystine, an amino acid that is absent in bovine erythrocyte carbonic anhydrase. Dithiothreitol greatly accelerated the rate of inactivation of the membrane-associated enzyme in SDS, so disulfide bonds appear to stabilize this enzyme. The specific CO2-hydrating activity was about half that of the erythrocyte enzyme. Acetazolamide inhibits the membrane-associated enzyme (Ki = 10 nM) nearly as well as the erythrocyte enzyme (Ki = 3 nM). Antibody to bovine erythrocyte carbonic anhydrase did not inhibit the membrane-associated enzyme. Other investigators have accumulated a good deal of evidence for carbonic anhydrase on the luminal surface of pulmonary capillaries. The enzyme described here appears to be a new isozyme whose properties are consistent with such a localization.     

Purification and characterization of lysosomal alpha-glucosidase secreted by eukaryote Tetrahymena

A large amount of lysosomal acid hydrolases was released into the medium by Tetrahymena pyriformis strain W during growth. An extracellular lysosomal acid alpha-glucosidase has been purified 500-fold with a 41% yield to homogeneity, as judged by polyacrylamide gel electrophoresis. It was found to be a glycoprotein and to consist of a single 110,000-dalton polypeptide chain. The carbohydrate content of the alpha-glucosidase was equivalent to 2.8% of the total protein content, and the oligosaccharide moiety was composed of mannose and N-acetylglucosamine in a molar ratio of 6.7:2. The optimal pHs for hydrolysis of maltose and p-nitrophenyl-alpha-glucopyranoside, maltose, isomaltose, and glycogen were 1.1 mM, 2.5 mM, 33.0 mM, and 18.5 mg/ml, respectively. This purified enzyme appears to have alpha-1,6-glucosidase as well as alpha-1,4-glucosidase activity. Turanose has a noncompetitive inhibitory effect on the hydrolysis of maltose. The antibody raised against Tetrahymena acid alpha-glucosidase inhibited the hydrolysis of all substrates tested. These properties of Tetrahymena acid alpha-glucosidase were found to be similar to those of the human liver lysosomal alpha-glucosidase.     

An improved purification procedure for rat liver lysosomal phospholipase A1

A purification procedure is presented for the isolation of lysosomal acid phospholipase A1 (PLA1) from livers of non-pretreated rats, in a high yield and purity. The purification starts from a crude mitochondrial-lysosomal fraction. PLA1 is solubilised and subsequently purified by chromatography on concanavalin A-Sepharose, by chromatofocusing, and by gel filtration. After chromatofocusing, the enzyme is already purified 50200-fold with a yield of 50%, and after gel filtration 56600-fold with a yield of 7%. Purified PLA1 exhibits a specific activity of approx. 8.2 mumol phosphatidylethanolamine (preferred substrate) hydrolysed per min per mg protein, and upon chromatofocusing an apparent isoelectric point of 5.3 Gel filtration of purified PLA1 suggests a molecular mass of about 29 kDa, whereas in SDS-PAGE two proteins of 27 kDa and 55 kDa (mass ratio about 1/2) were visualised.     

Purification and characterization of acid trehalase from the yeast suc2 mutant

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.     

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.