The Association of Brain Hexokinase with Mitochondrial Membranes and its Functional Implications

Abstract

The intracellular location of brain hexokinase and the nature of its association with mitochondria were studied in calf and rat. Three different media were used in order to minimize possible artifacts in the homogenization process. Subcellular fractionation was followed testing, in addition to hexokinase, certain enzymes known as markers for the different cellular fractions. In the conditions used, hexokinase was almost entirely associated with mitochondria in calf, but only partially associated in rat. This is at variance with the rest of the enzymes involved in the glucose 6‐phosphate crossroads. The use of a “physiological saline” homogenization medium resulted in a considerable increase in the proportion of soluble hexokinase in rat brain. Repeated washings of mitochondria from calf brain released little hexokinase, but in rat brain a rapid release was observed. Upon submitochondrial fractionation by Sottocasa's method, a substantial amount of hexokinase appeared bound to the inner membrane in the case of calf brain. In rat brain, the evidence found also points to the binding of the greater part of hexokinase to the inner membrane. Brain mitochondria were found to be sensitive to atractyloside, which made it possible to locate mitochondrial bound hexokinase as external to the ATP barrier, both in calf and rat. These results indicate that mitochondrially bound hexokinase is only active on substrates located in the cytosol.