Date of Award

1-1-1987

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Abstract

Glucose-6-phosphatase, EC 3.1.3.9, and glucokinase, EC 2.7.1.2, catalyze opposing reactions in rat liver, reactions 1 and 2 below. The presence of these opposing reactions allows futile cycling between glucose and glucose-6-P to occur in liver which results in the net hydrolysis of ATP, reaction 3 below.(UNFORMATTED TABLE OR EQUATION FOLLOWS)$$\eqalignno{\hbox{Glucose + ATP} &{\buildrel \hbox{Glucokinase}\over\longrightarrow}\hbox{Glucose-6-P + ADP}&(1)\cr \hbox{Glucose-6-P + H$\sb2$O}&{\buildrel \hbox{Glucose-6-P'ase}\over\longrightarrow} \hbox{Glucose + Pi}&(2)\cr \hbox{ATP + H$\sb2$O}&\longrightarrow \hbox{ADP + Pi}&(3)\cr}$$(TABLE/EQUATION ENDS) In an attempt to answer the question, "To what extent is glucose-6-phosphatase involved in the regulation of (glucose-6-P) and futile cycling between hepatic glucose and glucose-6-P?", the following aspects of hepatic glucokinase and glucose-6-phosphatase were examined.First, the conditions and assay methods used to measure in vitro glucokinase activity were examined. This was done to determine the assay conditions which would give the maximum in vitro glucokinase activity with near-physiological (glucose). The greatest glucokinase activity, with physiological (glucose) was obtained using 2.5 mM dithioerythritol and 100 mM KCl, in the continuous assay with Leuconostoc mesenteroides glucose-6-P dehydrogenase with the cofactor ${\rm NAD}\sp+.$Secondly, the stopped-flow assay technique was used to estimate the functional glucose-6-phosphatase concentration, Eo. Using this technique changes in Eo in response to changes in metabolic state were measured. It was found that changes in the maximum reaction velocity, observed under various metabolic states, could be correlated with changes in Eo in all situations except the triamcinalone treated groups.Third, glucose-6-phosphatase activity of intact and disrupted microsomes were determined using physiological (glucose-6-P). This was done to determine if "physiological" glucose-6-phosphatase activity follows classic Michaelis-Menten kinetics with respect to (glucose-6-P). It was found that intact microsomal glucose-6-phosphatase activity followed sigmoid kinetics with respect to low, near-physiological (glucose-6-P). It was concluded from these studies that activation of intact microsomal glucose-6-phosphatase by physiologically significant (glucose-6-P) is important in the regulation of futile cycling between hepatic glucose and glucose-6-P.

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