Isolation and characterization of angiotensin converting enzyme from human kidney

Angiotensin converting enzyme [EC 3.4.15.1] was solubilized from the membrane fraction of human kidney cortex using trypsin and purified to homogeneity by DEAE-cellulose, hydroxylapatite and DEAE-Sephadex A-50 column chromatographies, preparative isoelectric focusing, and Sephadex G-200 gel filtration. The final recovery of the enzyme was 13.9%. The molecular weight of the enzyme was estimated to be 199,000 by a sedimentation equilibrium method. A value of 170,000 was obtained for the reduced and denatured enzyme by dodecylsulfate-polyacrylamide gel electrophoresis. The enzyme was a glycoprotein consisting of a single polypeptide chain with an isoelectric point of 5.10. Neutral sugar accounted for 13% per weight of the enzyme. The purified enzyme had a specific activity of 96.9 mumol/min/mg protein for hippurylhistidylleucine. The Km value, Kcat value and hydrolytic coefficient (Kcat/Km) of the enzyme for hippurylhistidylleucine were 2.0 mM, 545 s-1 and 273 mM-1 . s-1, respectively. Rabbit antibody against the human kidney converting enzyme inhibited the activities of the enzymes from human lung and serum as equally as that from human kidney, but not those from sheep, dog, or rat sera. The human kidney and lung converting enzymes were immunologically identical on double immunodiffusion analysis.     

Synthesis of maltotriose by maltase purified from rabbit kidney.

To clarify the enzyme participates in maltotriose synthesis, we purified maltase from rabbit kidney using 2-amino-2-hydroxymethylpropane-1,3-diol (Tris) affinity column chromatography. The purified enzyme possessed specific activity of 33.7 mumol/mg/min and estimated molecular weight of 350,000 dalton, as judged by SDS-polyacrylamide gel electrophoresis, comparable with those reported from rat kidney. Moreover this enzyme possessed not only maltase (maltose----glucose) but also amylomaltase (maltose----maltotriose) activity, and both activities were inhibited by Tris in a dose-dependent manner with similar IC50 values. From these results, we concluded that maltotriose was synthesized by maltase in vitro and that kidney maltase may participate in sugar metabolism in vivo.     

Rapid purification of dipeptidyl peptidase IV from rat liver plasma membrane

Dipeptidyl peptidase IV (EC 3.4.14.5) was solubilized from rat liver plasma membranes with sulphobetaine 14 and purified by successive affinity chromatography on Con A-Sepharose, wheat germ lectin-Sepharose and arginine-Sepharose columns. The specific activity of the final preparation was 49.4 mumol Gly-Pro p-nitroanilide/min per mg protein, representing a 1098-fold purification of the homogenate. SDS-polyacrylamide gel electrophoresis of the arginine-Sepharose eluate showed a single protein band with a molecular weight of 105,000. The isoelectric point was determined to be 3.9 under non-denaturing conditions with sulphobetaine 14. The preparation was free of post-proline cleaving enzyme. The content of aminopeptidase M was 0.2% of the total protein.     

人脑醛糖还原酶的纯化和一些基本性质

使用DEAE纤维素柱层析、PBE-94层析聚焦、NADP~+-Sepharose 4B亲合层析及SephadexG-100凝胶过滤分离纯化了人脑醛糖还原酶。在DEAE层析中,用咪唑-HCI缓冲液替代了磷酸缓冲液,改善了分离效果。在聚丙烯酰胺及SDS聚丙烯酰胺凝胶电泳中,纯化的人脑醛糖还原酶均呈一条区带。它的pI为5.6,最适pH为6.5,分子量为36,000,底物特异性和氨基酸组成与其它哺乳动物的醛糖还原酶有相似性。开链式醛糖是醛糖还原酶的真正底物,它在开链式和半缩醛的平衡体系中占比例极小,因而推知醛糖还原酶对此底物有很高的K_(cat)和K_(cat)/K_m值,能有效地将它们还原成相应的醇。     

Purification and properties of a second enzyme catalyzing the splitting of carbon-mercury linkages from mercury-resistant Pseudomonas K-62.

An enzyme (splitting enzyme 2) which catalyzes the splitting of carbon-mercury linkage of arylmercury compounds was found in extracts of mercury-resistant Pseudomonas K-62. This enzyme was purified about 725-fold by treatment with streptomycin, precipitation with ammonium sulfate, and successive chromatography on Sephadex G-75 and diethylaminoethyl-cellulose. A purified preparation of the enzyme showed a single band in electrophoresis either on polyacrylamide or sodium dodecyl sulfate-containing polyacrylamide gels. The molecular weight of the enzyme was estimated to be 20,000 (determined by Sephadex G-75 gel filtration) 17,000 (determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis). The enzyme showed a Km of 180 micron and a Vmax of 3.1 mumol/min per mg for p-chloromercuribenzoic acid and a Km of 250 micron and a Vmax of 20 mumol/min per mg for phenylmercuric acetate. The optimum temperature and pH for the reaction were 40 degrees C and 5.0, respectively.     

The purification and characterization of human kidney L-arginine:glycine amidinotransferase

Human kidney L-arginine:glycine amidinotransferase (transamidinase) has been purified to a homogeneous state as defined by native and sodium dodecyl sulfate gel electrophoresis and by ultracentrifugation (sedimentation equilibrium) experiments. The four steps in the isolation procedure were chromatography with DEAE-cellulose, gel filtration with Sephadex G-150, chromatography with phenyl Sepharose, and high-pressure liquid chromatography with hydroxylapatite. The final product represented a 90-fold purification of the enzyme. Human kidney transamidinase is a dimer with a molecular mass of 89,000 Da and subunit masses of 44,000 Da. The Km for arginine and glycine were both 2.5 mM and the Vmax was 0.5 mumol ornithine/min/mg protein. The ultraviolet absorption spectrum, specific activity, and isoelectric points were determined for human kidney transamidinase. Multiple forms of the enzyme were obtained by isoelectric focusing. Human kidney transamidinase cross-reacted with polyclonal antibodies raised to rat kidney transamidinase. All of the properties of human kidney transamidinase that we have examined were similar to those of rat kidney transamidinase. A close evolutionary relationship between the rat and human kidney transamidinase is suggested.     

Purification of a human urinary carboxypeptidase (kininase) distinct from carboxypeptidases A, B, or N

A carboxypeptidase which cleaves basic C-terminal amino acids from peptides was purified from concentrated human urine by a three-step procedure: chromatography on Affi-Gel Blue, arginine-Sepharose affinity chromatography, and gel filtration by HPLC on a TSK-G3000SW column. Urinary carboxypeptidase was purified 406-fold with an 11% yield and a specific activity of 49 mumol/min/mg with benzoylglycylargininic acid as substrate. It migrated as a single band of Mr 75,700 in polyacrylamide gel electrophoresis with sodium dodecyl sulfate. It cleaved benzoylglycylarginine, benzoylglycyllysine, benzoylglycylargininic acid, benzoylalanyllysine, and benzoylphenylalanyllysine at different relative rates than human plasma carboxypeptidase N, the Mr 48,000 active subunit of carboxypeptidase N or human pancreatic carboxypeptidase B. Urinary carboxypeptidase did not hydrolyze benzoylglycylphenylalanine, a substrate of carboxypeptidase A, but readily cleaved bradykinin with a Km of 46 microM and a Kcat of 32 min-1. Its activity was enhanced by CoCl2 and inhibited by cadmium acetate, o-phenanthroline, or DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid. The enzyme had a pH optimum of 7.0 and its activity dropped at pH 6.0 by 60%. It was stable for at least 2 h at 37 degrees C (pH 8.0) but was unstable at room temperature below pH 4.5. The molecular weight, electrophoretic mobility, and activity of urinary carboxypeptidase was not affected by trypsin. The effect of pH and stability further distinguished the urinary carboxypeptidase from other human carboxypeptidases. Urinary carboxypeptidase was immunologically distinct from carboxypeptidase N when analyzed by the "Western blot" technique. Thus, human urine contains a basic carboxypeptidase, different from known carboxypeptidases, which may be released into the urine by the kidney. Here it could inactivate kinins and other peptides containing a basic C-terminal amino acid.     

Ketopantoyl-lactone reductase from Candida parapsilosis: purification and characterization as a conjugated polyketone reductase

Ketopantoyl-lactone reductase (2-dehydropantoyl-lactone reductase, EC 1.1.1.168) was purified and crystallized from cells of Candida parapsilosis IFO 0708. The enzyme was found to be homogeneous on ultracentrifugation, high-performance gel-permeation liquid chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of the native and SDS-treated enzyme is approximately 40,000. The isoelectric point of the enzyme is 6.3. The enzyme was found to catalyze specifically the reduction of a variety of natural and unnatural polyketones and quinones other than ketopantoyl lactone in the presence of NADPH. Isatin and 5-methylisatin are rapidly reduced by the enzyme, the Km and Vmax values for isatin being 14 microM and 306 mumol/min per mg protein, respectively. Ketopantoyl lactone is also a good substrate (Km = 333 microM and Vmax = 481 mumol/min per mg protein). Reverse reaction was not detected with pantoyl lactone and NADP+. The enzyme is inhibited by quercetin, several polyketones and SH-reagents. 3,4-Dihydroxy-3-cyclobutene-1,2-dione, cyclohexenediol-1,2,3,4-tetraone and parabanic acid are uncompetitive inhibitors for the enzyme, the Ki values being 1.4, 0.2 and 3140 microM, respectively, with isatin as substrate. Comparison of the enzyme with the conjugated polyketone reductase of Mucor ambiguus (S. Shimizu, H. Hattori, H. Hata and H. Yamada (1988) Eur. J. Biochem. 174, 37-44) and ketopantoyl-lactone reductase of Saccharomyces cerevisiae suggested that ketopantoyl-lactone reductase is a kind of conjugated polyketone reductase.     

Purification and characterization of human placental microsomal aminopeptidase: immunological difference between placental microsomal aminopeptidase and pregnancy serum cystyl-aminopeptidase

Human placental microsomal aminopeptidase (microsomal PAP) was purified 3,880-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100 and also trypsin digestion, and subjected to zinc sulfate fractionation, chromatographies with DE-52, hydroxylapatite, Sephacryl S-300 and lentil lectin-Sepharose 4B, and finally affinity chromatography with bestatin-Sepharose 4B. Microsomal PAP was separated from aminopeptidase A (AAP) by affinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 220,000 by high-performance liquid chromatography with an aqueous gel column. The purified enzyme gave almost a single band with a molecular mass of 140,000 by sodium dodecyl sulfate (SDS) gel electrophoresis. The isoelectric point of the enzyme was 5.2. The purified enzyme was most active at pH 8.0 with L-leucine-p-nitroanilide as substrate; the Km value for this substrate was 1.1 mmol/l. The microsomal PAP was immunologically different from the pregnancy serum cystyl aminopeptidase (serum PAP).     

Purification and characterization of an enkephalin aminopeptidase from rat brain membranes

A membrane-bound aminopeptidase was purified from rat brain, and its activity was assayed by high-pressure liquid chromatography with Met-enkephalin as the substrate. The enzyme was extracted with 1% Triton X-100 and purified by chromatography, successively on DEAE-Sepharose CL-6B, Bio-Gel HTP, and Sephadex G-200 columns. The overall purification was about 1200-fold, with 25% yield. The purified enzyme showed one band on disc gel electrophoresis and two bands on sodium dodecyl sulfate electrophoresis with molecular weights of 62 000 and 66 000. The aminopeptidase has a pH optimum of 7.0, a Km of 0.28 mM, and a Vmax of 45 mumol (mg of protein)-1 min-1 for Met-enkephalin. It releases tyrosine from Met-enkephalin, but it does not split the byproduct. It does not hydrolyze gamma- or beta-endorphin, or dynorphin, but it does hydrolyze neutral and basic aminoacyl beta-naphthylamides. The enzyme is inhibited by the aminopeptidase inhibitors amastatin, bestatin, and bestatin-Gly. Its properties, such as its subcellular localization, substrate specificity, pH optimum, and molecular weight, distinguish it from leucine aminopeptidase, aminopeptidase A, aminopeptidase B, aminopeptidase M, and the soluble aminopeptidase for enkephalin degradation.     

Purification and characterization of alpha-N-acetylgalactosaminidase from skipjack liver

By means of a simple procedure involving two gel filtrations and an ion-exchange chromatography, alpha-N-acetylgalactosaminidase was purified to an electrophoretically homogeneous form from skipjack liver, in which the enzyme is the dominant glycosidase. The final alpha-N-acetylgalactosaminidase preparation contained practically no other glycosidase activities except alpha-galactosidase activity, which amounted to 0.8% of the alpha-N-acetylgalactosaminidase activity and may be an intrinsic activity of the enzyme. The molecular weight of the enzyme was estimated to be 80,000 at pH 4.2 and 40,000 at pH 7.2 by molecular sieve chromatography, and to be 35,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 4 and was inactive above pH 7. These results suggest that skipjack alpha-N-acetylgalactosaminidase exists as an active dimer at acidic pH and as inactive monomer at neutral or alkaline pH. The enzyme efficiently liberated the N-acetylgalactosamine unit from ovine submaxillary glycoprotein which had been desialylated by neuraminidase. The Km value and maximum velocity were 4.28 mM and 409 mumol/min X mg for p-nitrophenyl alpha-N-acetylgalactosaminide, and 0.0543 mM and 1.19 mumol/min X mg for ovine submaxillary asialoglycoprotein.     

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.     

Uronic acid dehydrogenase from Pseudomonas syringae. Purification and properties.

1. Uronic acid dehydrogenase was purified to homogeneity. After a 338-fold purification a yield of 16% was achieved with a specific activity of 81 mumol NADH formed min-1 mg protein-1. 2. The purity of the enzyme was controlled by disc electrophoresis, sodium dodecylsulfate electrophoresis and ultracentrifugation. 3. A molecular weight of 60 000 was determined by gel chromatography and by ultracentrifugation. 4. The native enzyme is composed of two subunits, their molecular weight being 30 000 as estimated by sodium dodecylsulfate electrophoresis. The subunits as such are inactive. 5. The absorption spectrum with a maximum at 278 nm shows no evidence for a prosthetic group. 6. For catalytic activity no SH groups and no metals seem to be necessary. 7. The Michaelis constants determined with the pure enzyme are for glucuronic acid Km = 0.37 mM, galacturonic acid Km = 54 muM and NAD+ (with glucuronic acid) Km = 80 muM. 8. A weak reverse reaction could be observed with glucaric acid lactones at acidic pH. 9. NADH is competitive with NAD+. The inhibitor constant is Ki = 60 muM. 10. The NAD+ binding site seems to be of lower specificity than the uronic acid binding site.     

Purification and characterization of thermostable alpha-galactosidase from Ganoderma lucidum

Alpha-galactosidase was purified from a fresh fruiting body of Ganoderma lucidum by precipitation with ammonium sulfate and column chromatographies with DEAE-Sephadex and Con A-Sepharose. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. Its N-terminal amino acid sequence was similar to that of Mortierella vinacea alpha-galactosidase. The molecular mass of the enzyme was about 56 kDa by SDS-polyacrylamide gel electrophoresis, and about 249 kDa by gel filtration column chromatography. The optimum pH and temperature were 6.0 and 70 degrees C, respectively. The enzyme was fully stable to heating at 70 degrees C for 30 min. It hydrolyzed p-nitrophenyl-alpha-D-galactopyranoside (Km=0.4 mM) but hydrolyzed little o-nitrophenyl-alpha-D-galactopyranoside. It also hydrolyzed melibiose, raffinose, and stachyose. The enzyme catalyzed the transgalactosylation reaction which synthesized melibiose. The product was confirmed by various analyses.     

Purification and partial characterization of glycogen synthase kinase-3 from rabbit liver

Summary Glycogen synthase kinase-3 (GSK-3) was purified from rabbit liver to homogeneity by ultracentrifugation, ion-exchange chromatography on DEAE-cellulose, Cellulose phosphate, CM-Sephadex and Fast Protein Liquid Chromatography (FPLC) on Mono-S column. The enzyme was purified approximately 20,000 fold with an approximate 2% recovery. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis. GSK-3 is a monomeric enzyme with a molecular weight of 50,000–52,000 as derived from SDS-polyacrylamide gel electrophoresis and gel filtration. The purified enzyme was indeed a GSK-3 since it phosphorylated three sites, i.e., 3a, 3b, and 3c on liver glycogen synthase. GSK-3 incorporated up to 2.6 mol Pi/mol glycogen synthase subunit with a concomitant inactivation of glycogen synthase activity.     

Purification and properties of acyl/alkyl dihydroxyacetone-phosphate reductase from guinea pig liver peroxisomes

The peroxisomal acyl/alkyl dihydroxyacetone-phosphate reductase (EC 1.1.1.101) was solubilized and purified 5500-fold from guinea pig liver. The enzyme could be solubilized by detergents only at high ionic strengths in presence of the cosubstrate NADPH. Peroxisomes, isolated from liver by a Nycodenz step density gradient centrifugation, were first treated with 0.2% Triton X-100 to remove the soluble and a large fraction of the membrane-bound proteins. The enzyme was solubilized from the resulting residue by 0.05% Triton X-100, 1 M KCl, 0.3 mM NADPH, and 2 mM dithiothreitol in Tris-HCl buffer (10 mM) at pH 7.5. The enzyme was further purified after precipitating it by dialyzing out the KCl and then resolubilized with 0.8% octyl glucoside in 1 M KCl (plus NADPH and dithiothreitol). The second solubilized enzyme was purified to homogeneity (370-fold from peroxisomes) by gel filtration in a Sepharose CL-6B column followed by affinity chromatography on an NADPH-agarose gel matrix. NADPH-agarose was prepared by reacting periodate-oxidized NADP+ to adipic acid dihydrazide-agarose and then reducing the immobilized NADP+ with NaBH4. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed a single homogeneous band with an apparent molecular weight of 60,000. The molecular weight of the native enzyme was estimated to be 75,000 by size exclusion chromatography. Amino acid analysis of the purified protein showed that hydrophobic amino acid comprised 27% of the molecule. The Km value of the purified enzyme for hexadecyldihydroxyacetone phosphate (DHAP) was 21 microM, and the Vmax value in the presence of 0.07 mM NADPH was 67 mumol/min/mg. The turnover number (Kcat), after correcting for the isotope effect of the cosubstrate NADP3H, was calculated to be 6,000 mol/min/mol of enzyme, assuming the enzyme has a molecular weight of 60,000. The purified enzyme also used palmitoyldihydroxyactone phosphate as a substrate (Km = 15.4 microM, and Vmax = 75 mumol/min/mg). Palmitoyl-DHAP competitively inhibited the reduction of hexadecyl-DHAP, indicating that the same enzyme catalyzes the reduction of both acyl-DHAP and alkyl-DHAP. NADH can substitute for NADPH, but the Km of the enzyme for NADH (1.7 mM) is much higher than that for NADPH (20 microM). The purified enzyme is competitively (against NADPH) inhibited by NADP+ and palmitoyl-CoA. The enzyme is stable on storage at 4 degrees C in the presence of NADPH and dithiothreitol.     

Purification and Characterization of Aminopeptidase from Euphausia superba

Krill aminopeptidase was purified about, 1,100-fold from an extract of Euphausia superba with DEAE-cellulose column chromatography, Toyopearl HW55, and hydroxyapatite column chromatography. The final preparation was electrophoretically homogeneous. The molecular weight was determined to be 140,000 by gel filtration and SDS-polyacrylamide disc gel electrophoresis. The optimum pH and optimum temperature were 8.4 and 45°C respectively. Krill aminopeptidase was inhibited by EDTA, Hg^{+ +} and amastatin, and partially by bestatin, and was activated by Co ^{+ +}. Alanyl-p-nitroanilide was hydrolyzed faster than leucyl-p-nitroanilide. Alanyl peptides (di-, tri-, tetra- and hexa-alanyl peptide) were hydrolyzed very fast.

These results suggest that krill aminopeptidase is an alanine aminopeptidase which is activated by cobaltous ion.     

Purification and characterization of carboxypeptidase from terminally differentiated rat epidermal cells

A tissue carboxypeptidase-A-like enzyme was purified to apparent homogeneity from terminally differentiated epidermal cells of 2-day-old rats by potato inhibitor affinity chromatography followed by FPLC Mono Q column chromatography. The enzyme has an Mr of 35,000 as determined by SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It has a pH optimum of 8.5 for hydrolysis of benzyloxycarbonyl-Phe-Leu (Km = 0.22 mM, kcat = 57.9 s-1). The enzyme does not hydrolyze substrates with Arg, Lys and Pro at the C-terminal and Pro at the penultimate position. Angiotensin I was effectively hydrolyzed (Km = 0.06 mM, kcat = 6.48 s-1) and produced both des-Leu10-angiotensin I and angiotensin II. The enzyme activity, relatively stable at 4 degrees C and pH 8.0-10.5, was inactivated at pH values higher than 12.0 and lower than 5.0 or at 65 degrees C for 10 min. Inhibitor profiles of the epidermal enzyme also differed slightly from those of tissue carboxypeptidase A of pancreatic or mast cell origin.     

Purification and partial characterization of a Nocardia brasiliensis extracellular protease.

Nocardia brasiliensis possess proteolytic activities that can be readily detected in a variety of media. In a modified formulation of a growth medium originally used for Streptomyces aureofaciens, N. brasiliensis was found to secrete proteolytic enzymes, one of which was capable of hydrolyzing casein. This enzyme was purified to homogeneity from cell-free culture filtrates of N. brasiliensis. The purification procedure included ion-exchange chromatography on carboxymethyl-Sepharose, gel filtration on Sephadex G-100, and affinity chromatography, using a hemoglobin-Sepharose resin. The molecular weight of the N. brasiliensis protease was found to be 25,000 by gel filtration and 35,000 by sodium dodecyl sulfate-discontinuous gel electrophoresis. The enzyme is inhibited by o-phenanthroline and 8-hydroxyquinoline-5-sulfonic acid but is not affected by EDTA. Average values for its kinetic parameters were 0.288 mumol of hemoglobin solubilized per min per mg of enzyme for Vmax and 0.76 mM for Km, using hemoglobin as the substrate.     

Purification and characterization of alpha-galactosidase from watermelon

An alpha-galactosidase [EC 3.2.1.22] was isolated from the fruit of the watermelon, Citrullus battich. The enzyme was purified by procedures including extraction, ammonium sulfate precipitation, and chromatographies on DEAE-Sephadex, CM-Sephadex and Sephadex G-100. The final preparation was found to be fairly homogeneous on disc and SDS-polyacrylamide gel electrophoresis, and sufficiently free from other glycosidase activities. The molecular weight of the enzyme was estimated to be 45,000 by Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 4.5 for natural substrates and at 5.9 for artificial substrates. The enzyme liberates the alpha-galactose units from oligosaccharides of the raffinose series and ceramide trihexoside, and the hemagglutination-inhibiting activities of human ovarian cyst B-glycoprotein and blood group B-type ghosts were abolished by the enzyme.