title: Glycosidases of Ehrlich ascites tumor cells and ascitic fluid--purification and substrate specificity of alpha-N-acetylgalactosaminidase and alpha-galactosidase: comparison with coffee bean alpha-galactosidase.
abstract: Ehrlich ascites tumor cells and ascitic fluid were assayed for glycosidase activity. alpha-Galactosidase and beta-galactosidase, alpha- and beta-mannosidase, alpha-N-acetylgalactosaminidase, and beta-N-acetylglucosaminidase activities were detected using p-nitrophenyl glycosides as substrates. alpha-Galactosidase and alpha-N-acetylgalactosaminidase were isolated from Ehrlich ascites tumor cells on epsilon-aminocaproylgalactosylamine-Sepharose. alpha-Galactosidase was purified 160,000-fold and was free of other glycosidase activities. alpha-N-Acetylgalactosaminidase was also purified 160,000-fold but exhibited a weak alpha-galactosidase activity which appears to be inherent in this enzyme. Substrate specificity of the alpha-galactosidase was investigated with 12 substrates and compared with that of the corresponding coffee bean enzyme. The pH optimum of the Ehrlich cell alpha-galactosidase centered near 4.5, irrespective of substrate, whereas the pH optimum of the coffee bean enzyme for PNP-alpha-Gal was 6.0, which is 1.5 pH units higher than that for other substrates of the coffee bean enzyme. The reverse was found for alpha-N-acetylgalactosaminidase: the pH optimum for the hydrolysis of PNP-alpha-GalNAc was 3.6, lower than the pH 4.5 required for the hydrolysis of GalNAc alpha 1,3Gal. Coffee bean alpha-galactosidase showed a relatively broad substrate specificity, suggesting that it is suited for cleaving many kinds of terminal alpha-galactosyl linkages. On the other hand, the substrate specificity of Ehrlich alpha-galactosidase appears to be quite narrow. This enzyme was highly active toward the terminal alpha-galactosyl linkages of Ehrlich glycoproteins and laminin, both of which possess Gal alpha 1, 3Gal beta 1,4GlcNAc beta-trisaccharide sequences. The alpha-N-acetylgalactosaminidase was found to be active toward the blood group type A disaccharide, and trisaccharide, and glycoproteins with type A-active carbohydrate chains.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
authors.forename: F
authors.initials: F
authors.lastname: Yagi
authors.forename: A E
authors.initials: AE
authors.lastname: Eckhardt
authors.forename: I J
authors.initials: IJ
authors.lastname: Goldstein
mesh_terms: D000818:Animals; D001201:Ascites / Q000201:enzymology; D002240:Carbohydrate Sequence; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D002846:Chromatography, Affinity; D003069:Coffee; D005696:Galactosidases / Q000378:metabolism*; D006596:Hexosaminidases / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity; D000519:alpha-Galactosidase / Q000302:isolation & purification / Q000378:metabolism*; D048809:alpha-N-Acetylgalactosaminidase
chemical_list: D003069:Coffee; D005696:Galactosidases; D006596:Hexosaminidases; D000519:alpha-Galactosidase; D048809:alpha-N-Acetylgalactosaminidase