Model for the interaction of membrane-bound substrates and enzymes. Hydrolysis of ganglioside GD1a by sialidase of neuronal membranes isolated from calf brain.

Microsomes and synaptosomes isolated from calf brain contain a sialidase which cleaves ganglioside substrates. The hydrolysis of [3H]ganglioside GD1a by the membrane-bound enzyme has been studied under various conditions. The reaction rate decreased with increasing ionic strength in the incubation mixture, and was progressively enhanced by increasing concentrations of the primary alcohols n-pentanol to n-octanol. This stimulation correlates quantitatively with an increase in membrane 'fluidity' caused by these alcohols as measured by fluorescence depolarization employing 1,6-diphenyl-1,3,5-hexatriene as probe. The dependence of the reaction rate on the amount of enzyme in the incubation mixture was linear only with water-soluble substrates but not with the lipophilic ganglioside substrate. Evidence is presented that lipophilic substrate and enzyme interact mainly within the plane of the membrane presumably by lateral diffusion. Taking this into consideration Michaelis-Menten theory was modified accordingly. As predicted, apparent Km values increased linearly with the amount of membrane-bound enzyme added and decreased with the concentration of n-hexanol in the incubation mixture. In the presence of varying n-hexanol concentrations the apparent Km-value decreased with increasing membrane 'fluidity', as measured by fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene. On the other hand, as expected, V values were not affected by membrane 'fluidity' and increased linearly with the amount of membrane protein.