Brain hexokinase binds reversibly to the outer mitochondial membrane, with the relative distribution of the enzyme between bound and solubilized forms being governed by cellular concentrations of specific metabolites (glucose 6-phosphate probably being the most influential). This distribution is sensitive to the status of glucose metabolism in the cells, with increased proportions of bound enzyme found during periods of increased glycolytic activity. Variation in intracellular distribution is thought to represent a mechanims for regulation of catalytic activity, with the bound form of the enzyme being more active than the solubilized. The proposed work will focus on two major aspects of this phenomenon. First, immunohistochemical electron microscopic methods have been developed and, based on preliminary observations, may permit estimation of the soluble-particulate distribution of hexokinase in individual cells. Since, as noted above, this distribution is sensitive to relative glycolytic rate, the energy status in individual cells may be assessed by this method. A second objective of the proposed work will be to elucidate the molecular basis for the specific interaction between hexokinase and the outer mitochondrial membrane. A hexokinase-binding protein has been purified from the outer mitochondrial membrane. Preliminary studies have also suggested that it is the N-terminal region of the enzyme that is primarily involved in interaction with the membrane. Chemical and physical characterization of the hexokinase-binding protein and the N-terminal sequence of the enzyme may provide a more detailed understanding of the basis for this specific recognition and binding.
