Because of its strategic position at the termini of the two major pathways of glucose production in the liver and kidneys, the multicomponent glucose-6-phosphatase system is a logical site to target therapeutic agents intended to restrict hepatic and renal glucose production in patients with diabetes mellitus. The present proposal is designed to provide biochemical characterizations of the processes that comprise and control the glucose-6-phosphatase system. The specific objectives are (1) to resolve, purify, and characterize the individual components of the system, and (2) to identify and characterize the mechanism(s) that operate to exert short-term control over glucose-6-P hydrolysis in liver. Approaches to purifying the translocases will include affinity chromatography in attempts to exploit their differential reactivity with thiol reagents and stilbene derivatives. The characterization will include determinations of molecular weight, amino acid compositions, phospholipid dependencies, hydrophobic and hydrophilic domains, and definitions of the transport mechanism. Strategies for purifying the enzyme stress the use of specific stabilizing agents to permit manipulations that otherwise cause inactivation. Emphasis will be placed on identifying and assessing the physiologic role of a low molecular-weight compound that appears to confer stability on the enzyme in situ. Investigations of the mechanism of acute regulation will begin with an examination of two metabolic systems: (a) the activation by glucagon of glucose-6-phosphatase in fetal guinea pig livers at day 50 of gestation, and (b) the rapid inhibition of hepatic glucose-6-phosphate hydrolysis induced by acute, mild hyperglycemia in fasted rats. These studies will include (a) determinations of whether responses induced in intact livers are expressed as stable alterations in the kinetic characteristics of the system in cell-free systems (e.g. isolated microsomes), (b) screening of concentrated extracts from control and treated livers for regulatory agents, and (c) a consideration of the possible differential control of the glucose-6-phosphatase system in the granular and agranular regions of the endoplasmic reticulum. It is hoped that these studies will suggest new and innovative approaches to the clinical management of hyperglycemia in the diabetic patient.
