Effects of cetyltrimethylammonium bromide on catalytic properties of kidney microsomal glucose-6-phosphatase, inorganic pyrophosphate-glucose phosphotransferase and inorganic pyrophosphatase activities

Abstract

The modifying effects of the cationic detergent cetyltrimethylammonium bromide (CTAB) on catalytic properties of kidney microsomal d- glucose-6-P phosphohydrolase (EC 3.1.3.9) and associated inorganic pyrophosphatase and PP 1-glucose phosphotransferase activities have been studied. Initial reaction velocities, apparent Michaelis constant values and apparent maximal reaction velocity values were determined at various H + concentrations with microsomal preparations which had been supplemented with the detergent to a final concentration (w/v), of 0.00, 0.05, 0.10, 0.20 or 0.30% 15 min prior to assay. Measurements of initial reaction velocities at various assay pH values indicated that CTAB treatment produced a shift in pH optimum of glucose-6-P phosphohydrolase activity towards acid pH values and in that of inorganic pyrophosphatase and phosphotransferase activities in the direction of neutrality. Both on the basis of initial reaction velocity values and apparent maximal reaction velocity values extrapolated to infinite substrate concentrations, lower concentrations of CTAB (0.05 and 0.10%, w/v) were found to elevate all activities, while higher concentrations (0.20 and 0.30%, w/v) were less effective in this respect or actually inhibited. In contrast with these polyphasic effects of the detergent on velocity values, apparent Michaelis constant values for all phosphate substrates, assayed at pH 4.5, 5.5, and 6.5, all decreased progressively as CTAB concentrations were increased from 0.05% to 0.30% (w/v). Plots (see J. S. Friedenwald and G. D. Meangwyn-Davies, in W. D. McElroy and B. Glass, The Mechanism of Enzyme Action, Johns Hopkins Press, Baltimore 1954, p. 180), of reciprocals of apparent Michaelis constant values for these compounds versus CTAB concentrations were linear, supporting the concept that CTAB acts as a “coupling” activator at lower concentrations and as a noncompetitive inhibitor at higher concentrations. Apparent Michaelis constants for PP i—0.3–0.4 mM with 0.30% (w/v) CTAB (pH 5.5)—are the lowest such values yet reported. The apparent Michaelis constant value for glucose in the phosphotransferase reaction, in contrast, was only modestly changed by CTAB treatment of microsomes. Several alternative mechanistic interpretations are considered briefly.