Characterization of hexokinase isoenzyme types I and II in ascites tumor cells by an interaction with mitochondrial membrane

Summary

A difference was observed in the intracellular distribution between type I and II hexokinases in Ehrlich-Lettre hyperdiploid ascites tumor cells (ELD cells). Experiment of the rebinding to the mitochondria for either each or mixture of the partially purified preparations of the two types of hexokinase indicated that the accepting site on the mitochondrial membrane was common for both types. Mild treatment of the two isoenzymes with chymotrypsin resulted in loss of the binding ability to mitochondria without change in the catalytic activity. It was deduced from these results that the essential region in the two types of hexokinase to interact with mitochondria, which was cleaved by chymotrypsin, was the same or near-similar.

Secondly, rebinding to and releasing from mitochondria were examined for the two hexokinase isoenzymes in the presence of various factors affecting the interaction between hexokinase and mitochondria, such as divalent cations, glucose 6-phosphate, and Pi. In the absence of divalent cations, about a half of the type I isoenzyme was bound to mitochondria, whereas almost no type II was bound. A difference was also seen between the two types in the concentration of divalent cations required for the saturation of the binding. A more marked difference was observed in the effect of Pi either alone or in combination with glucose 6-phosphate on the activity and binding ability of the two hexokinases. For type I isoenzyme, Pi relieved both inhibitory and releasing effects of glucose 6-phosphate. On the contrary, for type II, Pi had no such a modulating effect on the releasing action of glucose 6-phosphate, and had the inhibitory effect for itself on the enzyme activity.

From these results, it is likely that the difference in the intracellular distribution between type I and II hexokinases in ELD cells is due to the difference in their catalytic regions in the reaction with these ligands, which would induce the structural change in the region responsible for the binding to mitochondria.