An examination of the in vivo distribution of brain hexokinase between the cytosol and the outer mitochondrial membrane

Abstract

These studies addressed the question of the in vivo distribution of rat brain hexokinase (HK), and whether physiologically relevant changes in the glycolytic rate are accompanied by changes in the distribution of HK. Homogenates of fresh tissue showed only 11–15% of the overt (assayable without added detergent) HK to be soluble (found in high-speed centrifugation supernatant fractions) when homogenization was begun within 15–20 s of sacrifice. Freeze-blown rat brain tissue also was used, coupled with a new technique wherein it was homogenized as it thawed in a buffered sucrose solution containing 1 m m EDTA. In tissue sampled 15 min (anesthetized) or 60 min (waking) after ip Nembutal injection (40 mg/kg), 23% of the overt HK and 79% of the total lactate dehydrogenase were soluble. The average phosphocreatine content of these and similar homogenates had decreased only 23% from in vivo levels, while ATP had decreased by 65%, due to the combined effects of a high level of endogenous ATPase, chelation of Mg 2+ by EDTA, and the greater stability of MgATP 2− relative to MgADP 1−. These data indicated that the tissue experienced, at most, the equivalent of 6 s of complete ischemia prior to the completion of homogenization. Synaptosomes derived from rat and chicken cerebra were incubated at 37 °C in a physiological salt solution containing 10 m m glucose. Addition of veratridine has been shown to stimulate glycolysis and oxidative phosphorylation two- to threefold (H. T. Kyriazi and R. E. Basford (1986) J. Neurochem., in press) , but did not alter the HK distribution, as 21% was found in the supernatant fractions of both control and veratridine-stimulated synaptosomes treated with digitonin. These results indicate that in brain tissue, large net movements of HK on and off the outer mitochondrial membrane do not occur, and thus play no role in the regulation of glycolysis.