Studies on glucosaminidase. 3. Testicular N-acetyl-β-glucosaminidase and N-acetyl-β-galactosaminidase

Publisher Website
Google Scholar
Abstract
P-Nitrophenyl N-acetylgalactosaminide has been used as a substrate to measure the N-acetylgalactosaminidase activity of a ram-testis extract. The photolysis of p-nitrophenyl N-acetylglucosaminide, which occurs in daylight after the enzymic hydrolysis has been stopped by adding sodium carbonate, was overcome by the use of borate buffer, pH 9.8. The presence of 0.01% of albumin or 0.02% of Triton X-100 in the enzymic solutions prevented a loss of specific activity oh dilution of the enzyme. No separation of the activities of N-acetylglucosaminidase and N-acetylgalactosaminidase was achieved by fractionation with ammonium sulphate or calcium phosphate gel, or on partial heat inactivation. The Ki values of the competing substances N-acetylglucosamine, 2-acetamido-2-deoxygluconolactone and phenyl N-acetylglucosaminide and of the corresponding galactosamine derivatives were measured. For each substance the value obtained with p-nitrophenyl N-acetylglucosaminide as substrate was almost the same as that obtained with p-nitrophenyl N-acetylgalactosaminide. The competition of p-nitrophenyl N-acetylglucosaminide and p-nitrophenyl N-acetylgalactosaminide for one enzyme site was confirmed by an experiment with mixtures of these substrates.