Model for the Interaction of Membrane‐Bound Substrates and Enzymes

Abstract

Microsomes and synaptosomes isolated from calf brain contain a sialidase which cleaves ganglioside substrates. The hydrolysis of [3H]ganglioside GDla 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-l,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 K,,, 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 K,-value decreased with increasing membrane 'fluidity', as measured by fluorescence depolarization of 1,6-diphenyl-l,3,5-hexatriene. On the other hand, as expected, I/ values were not affected by membrane 'fluidity' and increased linearly with the amount of membrane protein.Biological membranes contain enzymes acting on phospholipids and sphingolipids which are constituents of these membranes. During the investigation of two such systems we recognized that the reaction rate was highly dependent on the physical state of the membrane: degradation of sphingomyelin by neutral sphingomyelinase and of ganglioside GDI a by sialidase in neuronal membranes was greatly stimulated by general anesthetics which increase membrane 'fluidity' [ 1,2].Centrifugation studies [1] demonstrated that exogenous lipid substrates are adsorbed to or inserted into the membranes. A model was proposed suggesting an interaction between substrate and enzyme within the membrane by lateral diffusion [l]. Fluorescence depolarization studies with the probe 1,6-diphenyl-I ,3,5-hexatriene indicated a qualitative correlation between increase of membrane 'fluidity and stimulation of lipid degradation in the presence of general anesthetics. In the case of halothane this correlation was demonstrated at clinical concentrations [2,3]. In this paper a quantitative correlation between membrane 'fluidity' and the stimulation of degradation of ganglioside GDla employing the homologous series of primary alcohols will be presented.Attempts to determine kinetic parameters for the system mentioned above met with systematic difficulties, such as nonlinear protein-dependence and strong variations of the Dedicated to Professor Horst Jatzkewitz on the occasion of his 70th birthday.

Influence of major structural features of tocopherols and tocotrienols on their ω-oxidation by tocopherol-ω-hydroxylase

“…A reduction in membrane fluidity induced by cholesterol enrichment or phospholipid modification inhibits the activity of CYP7A and CYP3A (50). Conversely, an increase in fluidity induced by n-octanol enhances the activity of membrane sialidase and reduces K m with no effect on V max (51). In contrast, a-TOH increases the V max of the metabolism of g-TOH with no effect on K m .…”

Partial Characterization and Enrichment of a Membrane‐Bound Sialidase Specific for Gangliosides from Human Brain Tissue

“…Tn the presence of octylglucoside, the enzyme exhibited high affinity for the substrate ganglioside G,,, with an apparent K,,, of 5 pM. However, the presence of detergent creates highly artificial conditions, as the substrate is present as a inicelle mixed with detergent rather than embedded in a membrane [32]. Using ganglioside G,, as substrate, the pHactivity profile revealed a broad maximum around pH 5.5 that was distinct from the curve determined in the homogenate, where the overall maximum was around pH 4.…”

Combinatorial Ganglioside Biosynthesis

“…In addition, membrane-bound glycosyltransferases interact with their membrane-bound glycolipid substrates by diffusion within the two-dimensional plane of the lipid bilayer. This explains the observation that reaction rates can become independent of the reaction volume and may obey two-dimensional enzyme kinetics (17,18).…”