Studies on the substrate specificity of hexosaminidase A and B from liver

β-N-Acetylhexosaminidase A and B were partially purified from normal human liver using DEAE-cellulose column chromatography. Hexosaminidase B was also purified from the livers of patients who had died of Tay-Sachs disease. The hexosaminidase fractions were tested for their ability to hydrolyze the amino sugar moiety of synthetic substrates and of three amino sugar-containing glycolipids, GA2, globoside, and GM2. Hexosaminidase A and B had similar ability to catalyze the hydrolysis of synthetic N-acetylglycosaminides. The hexosaminidase B from the liver of a Tay-Sachs disease patient had a slightly higher Michaelis constant toward the same substrates as the control hexosaminidase B. Radioactively labeled GA2, N-acetyl-[1-14C] galactosaminyl (β 1 → 4)-galactosyl (β 1 → 4)-glucosyl (β 1 → 1)-ceramide was prepared biosynthetically. Hexosaminidase A and B from normal liver had identical Michaelis constants for the hydrolysis of GA2; whereas, the hexosaminidase B from a Tay-Sachs disease patient possessed a higher Michaelis constant. Globoside was hydrolyzed by both hexosaminidase A and B, although hexosaminidase B has a significantly lower Michaelis constant than the hexosaminidase A. Under the conditions of incubation neither hexosaminidase A nor B catalyzed the hydrolysis of the N-acetylgalactosaminyl residue from ganglioside GM2. However, in the presence of a neuraminidase preparation from Clostridium perfringens plus hexosaminidase A or B at pH 3.8 there was significant enzymatic hydrolysis of GM2 to ceramide lactose. Both GM2 and globoside were found to be excellent competitive inhibitors of GA2 hydrolysis by hexosaminidase B. These results are discussed with respect to the accumulation of these glycolipids in two GM2 ganglioside-storage diseases.