Enzymatic Action of Sialidase of Vibrio cholerae on Brain Gangliosides above and below the Critical Micelle Concentration

Abstract

Abstract The activity of Vibrio cholerae sialidase was studied as a function of the physical state of ganglioside substrate. This study provides a model for the interaction of end group hydrolases with highly polar anionic complex lipids. Sialyl groups of gangliosides are hydrolyzed by Vibrio sialidase whether the substrate is in disperse or in micellar form. The action of Vibrio sialidase on gangliosides appears, in this regard, to differ from the reported action of other end group hydrolases on complex lipids. Ultracentrifugal analyses indicate that a substantial proportion of sialidase in aqueous buffer adheres to and can be precipitated with ganglioside micelles, but the enzyme remains in solution in the presence of monodisperse substrate, as does monodisperse substrate alone. The effect upon the activity of a large total surface area of susceptible sialyl groups in the aggregated ganglioside substrate seems to almost compensate for the lower number of diffusing particles for this form. Regular dependence of initial velocity on substrate concentration is observed for both the micellar and submicellar substrate. Km below the critical micelle concentration (1 x 10-4 m) is roughly one-fifth the apparent Km for the substrate above the critical micelle concentration (5 x 10-4 m). The effect of hydrogen ion concentration on initial reaction velocity differs for aggregated (micellar) and disperse (submicellar) substrate. Above the pH optimum, enzymatic activity toward both substrate forms decreases very moderately with increasing pH, and there is a small increase in Km. Below the pH optimum, Km remains relatively invariant with decreasing pH. Although the substrate is strongly anionic at all pH values studied, there is no evidence that combination with the substrate affects the degree of ionization of any catalytic group on the enzyme. Minor loss of enzymatic activity occurs with the submicellar substrate below the pH optimum. However, great loss is observed for the micellar substrate when pH is decreased below the optimum. Mathematical analysis of the pH effect suggests that no single titratable functional group on the enzyme is involved in its catalytic activity. A noncompetitive time-dependent inhibitory effect is exerted by the micellar substrate at high concentration. The initial velocity of the enzymatic hydrolysis of sialyl groups of gangliosides can be increased to double the highest value obtainable with pure substrate by the admixture of nonsialyl lipid, e.g. lecithin. For this lipid plus gangliosides, there is a critical mixing ratio of unity for optimal activation. Activation of the sialyl residues of the aggregated substrate may be the result of spacing by the nonsialyl lipids. Properties of gangliosides when tested as substrates for soluble Vibrio sialidase may reflect properties of the sialyl groups of these glycosphingolipids in native neuronal plasma membranes.