HEXOKINASE AND ENERGY METABOLISM IN THE BRAIN

本项目的总体目标仍然是理解 影响大脑己糖激酶功能的各种因素 在调节大脑能量代谢方面发挥重要作用。八个具体的 目的：1.测定结合己糖激酶的分布。 在外部的接触点和非接触点区域之间 线粒体膜，并可能与其存在的相关性 单体或四聚形式；2.确定是否分布 接触部位和非接触部位区域之间的己糖激酶影响 连接到线粒体内的三磷酸腺苷；3.测定 己糖激酶与脑内线粒体结合差异的基础 研究己糖激酶与腺嘌呤的偶联反应。 含有纯化的核苷酸的平面脂质双层膜中的核苷酸转运 线粒体孔蛋白；5.利用定点突变技术，确定 涉及到的特定氨基酸残基的功能重要性 己糖激酶的催化或调节功能，或在结合 酶对线粒体的作用；6.检测相互作用的功能作用 通过研究己糖激酶的N-末端和C-末端结构域之间的关系 嵌合己糖激酶酶的催化和调节特性 I型(脑)同工酶的C末端(催化)结构域被融合 利用II型和II型同工酶的N-末端(调节)结构域， 反之亦然；7.分离该基因的5‘侧翼区 大鼠己糖激酶的I型(脑)同工酶，并定义顺式元件和 参与调控该基因表达的反式作用因子； 发展神经细胞培养系统，其中己糖激酶水平 对影响葡萄糖利用率的慢性治疗作出反应， 并确定己糖激酶水平的调节机制。 这项工作的结果将为进一步深入了解分子 己糖激酶的催化和调节功能的基础， 其与脑内线粒体相互作用的代谢后果 其他组织，以及补充酶的机制 神经组织可能会因代谢的慢性变化而改变 状态。

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.