METABOLIC MODULATION OF HEXOKINASE ASSOCIATION WITH MITOCHONDRIA IN LIVING SMOOTH-MUSCLE CELLS

Hexokinase isoform I binds to mitochondria of many cell types. It has been hypothesized that this association is regulated by changes in the concentrations of specific cellular metabolites. To study the distrib ution of hexokinase in living cells, fluorophore-labeled functional he xokinase I was prepared. After microinjection into A7r5 smooth muscle cells, hexokinase localized to distinct structures identified as mitoc hondria. The endogenous hexokinase demonstrated a similar distribution with the use of immunocytochemistry. 2-Deoxyglucose elicited an incre ase in glucose 6-phosphate (G-6-P) and a decrease in ATP levels and di minished hexobinase binding to mitochondria in single cells. 3-O-methy lglucose elicited slowly developing decreases in all three parameters. In contrast, cyanide elicited a rapid decrease in both ATP and hexoki nase binding. Analyses of changes in metabolite levels and hexokinase binding indicate a positive correlation between binding and cell energ y state as monitored by ATP. On the other hand, only in the presence o f 2-deoxyglucose was the predicted inverse correlation between binding and G-6-P observed. Unlike the relatively large changes in distributi on observed with the fluorescent-injected hexokinase, cyanide caused o nly a small decrease in the localization of endogenous hexokinase with mitochondria. These findings suggest that changes in the concentratio ns of specific metabolites can alter the binding of hexokinase I to sp ecific sites on mitochondria. Moreover, the apparent difference in sen sitivity of injected and endogenous hexokinase to changes in metabolit es may reflect the presence of at least two classes of binding mechani sms for hexokinase, with differential sensitivity to metabolites.