The long-range goal of these studies is to better-understand the structural inter-relationships, metabolic roles, and bioregulation of the bifunctional, multicomponent, membrane bound hepatic glucose-6- phosphatase system (EC 3.1.3.9) under normal, fasted, diabetic, cancerous, and other pathologic conditions. Glucose-6-phosphatase is an organized system within the endoplasmic reticulum consisting of at least six components: The catalytic unit itself, a stabilizing protein, and four (or more) translocases responsible for transport of individual substrates and products to/from the sequestered catalytic unit. Attention will be focused on translocase """"""""T2beta"""""""", the transporter for product Pi and substrates PPi and carbamyl-P, more specifically on its response and underlying mechanism in Ehrlich ascites tumor-bearing mice. Answers will be sought to the question of why Pi appears not to be transported from the medium (cytosol) to the lumen of the endoplasmic reticulum even though total T2beta protein increases three-fold in ascites tumor-bearing mice. As a working hypothesis, it is proposed that two T2beta forms exist, i.e , """"""""T2beta"""""""" in normal control and """"""""T2beta'"""""""" in liver microsomes derived from tumor-bearing mice. Studies are proposed to 1) correlate the time-frame for change in T2beta with tumor development; 2) examine the inhibition kinetics with liver microsomes from humor-bearing and control mice to delineate sites of common and distinct interactions among glucose-6-P, PPi, Pi, carbamyl-P, and glucose, and tumor-related changes therein; 3) determine the specificity and rates of transport of carbamyl-P, PPi, and Pi into, and out of, microsomes derived from tumor-bearing and control mouse liver; and 4) check for multiple forms of translocase T2beta using Western immunoblotting, combined with PAGE without and with isoelectric focusing, and isolate and chemically characterize these separate forms. Experimentation will include a combination of immunochemistry, protein chemistry, enzymology (primarily kinetics), transport studies with a novel fast-sampling, rapid-filtration apparatus, and isotope techniques for study of protein phosphorylation/ dephosphorylation. These studies will give valuable new knowledge of how the gluconeo/genically-stressed tumor-bearing mouse adapts in an attempt to maintain normoglycemia and to conserve glucose-6-P for the liver's use. The studies may also define new foci for anticancer drugs. A better understanding of how components of the glucose-6-phosphatase system function individually and in concert, normally and under such stressed conditions as diabetes, glucocorticoid administration, and the presence of developing tumors, will be achieved.
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