HEXOKINASE AND ENERGY METABOLISM IN THE BRAIN

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The overall objective of this project continues to be an understanding of factors governing the function of brain hexokinase, an enzyme playing a major role in regulation of cerebral energy metabolism. Eight specific aims are proposed: 1. determine the distribution of bound hexokinase between contact site and non-contact site regions of the outer mitochondrial membrane, and possible correlation with existence in monomeric or tetrameric forms; 2. determine whether the distribution of hexokinase between contact site and non-contact site regions affects coupling to intramitochondrial compartments of ATP; 3. determine the basis for differences in binding of hexokinase to mitochondria in brains of various species; 4. investigate coupling of hexokinase to adenine nucleotide transport in planar lipid bilayers containing purified mitochondrial porin; 5. using site-directed mutagenesis, determine the functional importance of specific amino acid residues implicated in catalytic or regulatory functions of hexokinase, or in binding of the enzyme to mitochondria; 6. examine the functional role of interactions between the N- and C- terminal domains of hexokinase by studying the catalytic and regulatory properties of chimeric hexokinases in which the C-terminal (catalytic) domain of the Type I (brain) isozyme is fused with the N-terminal (regulatory) domain of the Type II and II isozymes, and vice versa; 7. isolate the 5'-flanking region of the gene for the Type I (brain) isozyme of rat hexokinase, and define cis elements and trans-acting factors involved in regulating expression of this gene; 8. develop neural cell culture systems in which hexokinase levels are responsive to chronic treatments affecting rates of glucose utilization, and determine the mechanism by which hexokinase levels are regulated. The results of this work will provide further insight into the molecular basis for the catalytic and regulatory functions of hexokinase, the metabolic consequences of its interaction with mitochondria in brain and other tissues, and the mechanism by which the enzyme complement of neural tissue may be altered in response to chronic changes in metabolic status.