GM1-Ganglioside and lactosylceramide β-galactosidase from rabbit brain: inhibitor and substrate competition studies

Abstract

A rabbit brain β-galactosidase catalyzes the hydrolysis of synthetic substrates and the natural substrates GM1-ganglioside, lactosylceramide, and asialo-GM1-ganglioside. γ-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, whereas GM1-ganglioside inhibited lactosylceramide hydrolysis. At low substrate concentrations (<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 chloromercuriphenyl sulfonic acid was different for each substrate.At 0.5 μM chloromercuriphenyl sulfonic 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.