Application of a Double Isotopic Labeling Method to a Study of the Interaction of Mitochondrially Bound Rat Brain Hexokinase with Intramitochondrial Compartments of ATP Generated by Oxidative Phosphorylation

γ-Labeled ATP was produced by rat brain mitochondria utilizing [32P]Pias substrate for oxidative phosphorylation. The32P/14C ratio of Glc-6-P produced by the endogenous mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) using [U-14C]Glc as substrate was determined as a function of time after initiation of oxidative phosphorylation. This same ratio was determined for Glc-6-P formed by added yeast hexokinase using extramitochondrial ATP as substrate. The specific activity of ATP formed by oxidative phosphorylation was manipulated either by initiating the reaction with labeledPiand subsequently adding excess unlabeledPior by initiating the reaction with unlabeledPiand introducing the labeled substrate at a later time. The32P/14C ratio of Glc-6-P formed by yeast hexokinase, reflecting the specific activity of ATP in the extramitochondrial space, was rapidly responsive to such manipulations, but the corresponding changes in the32P/14C ratio of Glc-6-P produced by the endogenous hexokinase were markedly different. The results are consistent with the view that mitochondrially bound hexokinase does not utilize extramitochondrial ATP as substrate but rather is functionally coupled to a discrete intramitochondrial compartment of ATP produced by oxidative phosphorylation.