Isolation and further characterization of bovine brain hexosaminidase C.
Hexosaminidase C (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) was partially purified from bovine brain tissue. The resulting preparation, free of its lysosomal counterparts, was used for the characterization of the enzyme and for further purification (lectin affinity chromatography, hydrophobic interaction chromatography, substrate-ligand affinity chromatography, ion-exchange chromatography, chromatography on activated thiol-Sepharose 4B). Only ion-exchange chromatography on DEAE-Sephacel appeared to improve the purity. The Michaelis constant was 0.46 mM for the substrate 4-methyl-umbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside. The enzyme was not inhibited by acetate or N-acetylgalactosamine. Inhibition by N-acetylglucosamine was competitive, with a Ki value of 8.0 mM. Inhibition by divalent metal ions increased in the order Fe less than Zn less than Cu. Dithiothreitol and beta-mercaptoethanol, at an optimum concentration of about 10 mM, stimulated the activity. The enzyme is apparently not a glycoprotein since it did not bind to various lectins, nor did sialidase change its isoelectric point.
D000118:Acetylglucosaminidase / Q000302:isolation & purification*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004229:Dithiothreitol / Q000494:pharmacology; D006596:Hexosaminidases / Q000302:isolation & purification*; D007525:Isoelectric Focusing; D007700:Kinetics; D009439:Neuraminidase / Q000378:metabolism
D002413:Cations, Divalent; D006596:Hexosaminidases; D009439:Neuraminidase; D000118:Acetylglucosaminidase; D004229:Dithiothreitol