Neutral beta-N-acetylhexosaminidases of rat brain. Purification and enzymatic and immunological characterization.
The two neutral beta-N-acetylhexosaminidases of rat brain have been purified by procedures involving extraction, concanavalin A-Sepharose, ammonium sulfate precipitation, DEAE-cellulose, hydroxyapatite, Sepharose 4B, and an affinity chromatography with 2-acetamido-N-(epsilon-aminocaproyl)-2-deoxy-beta-galactopyranosylamine bound to Sepharose. The neutral beta-N-acetylgalactosaminidase was purified to homogeneity at 2000-2500-fold purification over the post-concanavalin A fraction. It was specific to beta-N-acetylgalactosaminide and was inactive to glucosaminide. It appeared to possess extremely small but detectable activity to hydrolyze the terminal N-acetylgalactosamine residue from GM2, asialo-GM2, and globoside. The other enzyme, neutral N-acetylglucosaminidase, was 85-90% pure, judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was predominantly active toward the N-acetylglucosaminide substrate but hydrolyzed N-acetylgalactosaminide at 0.5% of the rate toward glucosaminide. The possibility of this very small galactosaminidase activity being due to contamination by the first enzyme could be excluded by several criteria. Three different criteria, gel filtration, acrylamide gel electrophoresis, and sucrose density gradient centrifugation, gave inconsistent results in the estimated molecular size of either enzyme. The two enzymes were, however, different in molecular size by any of the criteria. Precipitating antibody was produced in rabbits against the neutral beta-N-acetylgalactosaminidase. It specifically precipitated the enzyme but did not cross-react with the neutral beta-N-acetylglucosaminidase or the acid beta-N-acetylhexosaminidase A and B. Physiological substrates for these neutral hexosaminidases are yet to be clarified.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D006596:Hexosaminidases / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D051381:Rats; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D001619:beta-N-Acetylhexosaminidases
D007527:Isoenzymes; D013439:Sulfhydryl Reagents; D006596:Hexosaminidases; D001619:beta-N-Acetylhexosaminidases