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

gamma-labeled ATP was produced by rat brain mitochondria utilizing [P- 32]P-i as substrate for oxidative phosphorylation. The P-32/C-14 ratio of Glc-6-P produced by the endogenous mitochondrially bound hexokinas e (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) using [U-C-14]Glc as substrate was determined as a function of time after initiation of ox idative phosphorylation. This same ratio was determined for Glc-6-P fo rmed by added yeast hexokinase using extramitochondrial ATP as substra te, The specific activity of ATP formed by oxidative phosphorylation w as manipulated either by initiating the reaction with labeled P-i and subsequently adding excess unlabeled P-i or by initiating the reaction with unlabeled P-i and introducing the labeled substrate at a later t ime. The P-32/C-14 ratio of Glc-6-P formed by yeast hexokinase, reflec ting the specific activity of ATP in the extramitochondrial space, was rapidly responsive to such manipulations, but the corresponding chang es in the P-32/C-14 ratio of Glc-6-P produced by the endogenous hexoki nase were markedly different, The results are consistent with the view that mitochondrially bound hexokinase does not utilize extramitochond rial ATP as substrate but rather is functionally coupled to a discrete intramitochondrial compartment of ATP produced by oxidative phosphory lation. (C) 1995 Academic Press, Inc.