Quantitative aspects of relationship between glucose 6-phosphate transport and hydrolysis for liver microsomal glucose-6-phosphatase system. Selective thermal inactivation of catalytic component in situ at acid pH.

Studies of the thermal stability of rat liver glucose-6-phosphatase (EC 3.1.3.9) were carried out to further elevate the proposal that the enzymic activity is the result of the coupling of a glucose-6-P-specific translocase and a nonspecific phosphohydrolase-phosphotransferase. Inactivation was observed when micorsomes were incubated at mild temperatures between pH 6.2 and 5.6. The rate of inactivation increased either with increasing hydrogen ion concentration or temperature. However, no inactivation was seen below 15 degrees in media as low as pH 5 or at neutral pH up to 37 degrees. The thermal stability of the enzyme may be controlled by the physical state of the membrane lipids and the degree of protonation of specific residues in the enzyme protein. Microsomes were exposed to inactivating conditions, and kinetic analyses were made of the glucose-6-P phosphohydrolase activities before and after supplementation to 0.4% sodium taurocholate. The results support the postulate and the kinetic characteristics of a given preparation of intact microsomes are determined by the relative capacities of the transport and catalytic components. Before detergent treatment, inactivation (i.e. a decrease in Vmax) was accompanied by a decrease in Km and a reduction in the fraction of latent activity, whereas only Vmax was depressed in disrupted preparations. The possibility that the inactivating treatments caused concurrent disruption of the microsomal membrane was ruled out. It is concluded that exposures to mild heat in acidic media selectively inactivate the catalytic component of the glucose-6-phosphatase system while preserving an intact permeability barrier and a functional glucose-6-P transport system. Analyses of kinetic data obtained in the present and earlier studies revealed several fundamental mathematical relationships among the kinetic constants describing the glucose-6-P phosphohydrolase activities of intact (i.e. the "system") and disrupted microsomes (i.e. the catalytic component). The quantitative relationships appear to provide a means to calculate a velocity constant (VT) and a half-saturation constant (KT) for glucose-6-P influx. The well documented, differential responses of the rat liver glucose-6-phosphatase system induced by starvation, experimental diabetes, or cortisol administration were analyzed in terms of these relationships. The possible influences of cisternal inorganic phosphate on the apparent kinetic constants of the intact system are discussed.