Hydrolysis of GM1-ganglioside by human liver β-galactosidase isoenzymes

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Abstract
1. GM1-ganglioside, specifically tritiated in the terminal galactose, was hydrolysed by two forms of ‘acid’ methylumbelliferyl β-galactosidase isolated on gel filtration. 2. Identification of GM1-ganglioside β-galactosidase activity with the ‘acid’ methyl-umbelliferyl β-galactosidases was based on the following: coincident elution profiles on gel filtration; simultaneous inactivation by heat and other treatments; stabilization of both activities by chloride ions; mutual inhibition of hydrolysis by the two substrates. 3. The two isoenzymes (I) and (II) showed general requirements for a mixture of anionic and nonionic detergents in the hydrolysis of the natural substrate. 4. Isoenzyme (I) differed from (II) in molecular size, pH–activity profile, relative resistance to dilution and in sensitivity to various inhibitors. 5. The most significant difference between the isoenzymes is in substrate saturation kinetics: (I) was hyperbolic whereas (II) was sigmoid. The apparent Michaelis constants were 28μm for (I) and 77μm for (II). Isoenzyme (I) was insensitive to GM2-ganglioside whereas (II) was inhibited, consistent with the hypothesis that GM1-ganglioside (and its analogue) acts as modifier in isoenzyme (II) but not in (I). 6. Isoenzyme (I) was membrane-bound whereas (II) was soluble; the former probably represents isoenzyme (II) bound to membrane components, thereby becoming activated. 7. Membranes may serve a dual role in enzyme catalysis involving lipids: as a medium where both enzyme and substrate are effectively concentrated, and as actual activator of enzymes through binding of the latter to specific membrane components.

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