GM1-ganglioside and lactosylceramide beta-galactosidase from rabbit brain: inhibitor and substrate competition studies.
A rabbit brain beta-galactosidase catalyzes the hydrolysis of synthetic substrates and the natural substrates Gm1-ganglioside, lactosylceramide, and asialo-Gm1-ganglioside. gamma-D-Galactonolactone competitively inhibited hydrolysis of Gm1-ganglioside, lactosylceramide, and MU-galactoside with Ki values of 0.26 mM, 0.13 mM, and 0.77 mM, respectively. From activity plots comparing the degree of inhibition to the inhibitor concentration, a single binding site for each substrate was found. NP-Galactoside inhibited the hydrolysis of Gm1-ganglioside and lactosylceramide, where as Gm1-ganglioside inhibited lactosylceramide hydrolysis. At low substrate concentrations (less than 1 mM), Gm1-ganglioside was hydrolyzed effectively in the presence of NP-galactoside, but at higher concentrations hydrolysis of the latter was preferred. Chloromercuriphenylsulfonic acid and iodoacetate were effective inhibitors of the enzyme, but N-ethylmaleimide was not. The degree of inhibition with chloromercuriphenylsulfonic acid was different for each substrate. At 0.5 mugM chloromercuriphenylsulfonic acid, all activity towards NP-galactoside, 75% towards lactosylceramide, and 25% of the Gm1-ganglioside activity was lost. Two possible models are presented to explain these results. The data favour the presence of multiple active sites in the enzyme.
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000201:enzymology*; D002518:Ceramides / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D005696:Galactosidases / Q000378:metabolism*; D006017:Glycolipids / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D007700:Kinetics; D011485:Protein Binding; D011817:Rabbits; D013329:Structure-Activity Relationship
D002518:Ceramides; D006017:Glycolipids; D007461:Iodoacetates; D005696:Galactosidases; D005033:Ethylmaleimide