BRANCHED CHAIN AMINO ACID METABOLISM IN SKELETAL MUSCLE

人们对基本的 支链氨基酸（亮氨酸，异亮氨酸，缬氨酸），因为 这些代谢产物的营养、生化和临床意义。 支链氨基酸的身体浓度被调节为 这一点可以通过导致以下疾病的严重代谢后果来证明： 过量或不成比例的这些氨基酸。的第一步 分解代谢途径是由支链催化的转氨作用， 氨基转移酶以形成支链α-酮酸。 我们 实验室已经确定有两种转氨酶同工酶- - 线粒体（BCAT-m）和胞质（BCAT-c）。 作为我们目标的一部分， 定义这些蛋白质的生理作用，我们将进行 纯化酶的详细动力学分析。 为了理解 支链氨基酸之间的转氨作用，线粒体转运 和体内氮代谢，一种体外细胞代谢模型 将使用分离的线粒体开发，在未来， 细胞 转氨作用后，支链α-酮酸氧化 发生在线粒体中。 这些α-酮酸的运输（和 随后的代谢物）必须发生在线粒体内部 膜的 我们已经确定了一个特定的线粒体转运系统 对于支链α-酮酸，其与第二种α-酮酸分离， 单羧酸转运蛋白，丙酮酸载体。 然而，在动力学上， 这些转运蛋白非常相似 支链的纯化 α-酮酸转运蛋白导致了新的发现， 线粒体氨基转移酶（mitochondrial aminotransferase，BCAT-m）是一种双功能蛋白 催化支链α-酮酸的转运， 转氨作用 我们的目标是确定BCAT-m的作用机制 催化转运和活性位点的分子结构。 本研究将包括使用共价修饰试剂来鉴定 参与转运的氨基酸残基和转氨酶 BCAT-M的活动。 将启动实验以确定 支链氨基转移酶同工酶的晶体结构。 作为 我们的第三个目标是建立一个线粒体的分子模型， 我们将克隆丙酮酸的cDNA， 传送器。 这项研究应该提供新的信息的多样性 阴离子运输机制。

There has been considerable interest in catabolism of the essential branched chain amino acids (leucine, isoleucine, valine) because of the nutritional, biochemical and clinical significance of these metabolites. Body concentrations of branched chain amino acids are regulated as evidenced by the severe metabolic consequences of disorders that result in excess or disproportionate amounts of these amino acids. The first step in the catabolic pathway is transamination catalyzed by the branched chain aminotransferase to form the branched chain alpha-keto acids. Our laboratory has established that there are two transaminase isoenzymes- - mitochondrial (BCAT-m) and cytosolic (BCAT-c). As part of our goal of defining the physiological role of these proteins, we will conduct detailed kinetic analyses of the purified enzymes. To understand the link between branched chain amino acid transamination, mitochondrial transport and body nitrogen metabolism, an in vitro model of cellular metabolism will be developed using isolated mitochondria and, in the future, intact cells. After transamination, oxidation of the branched chain alpha-keto acids occurs in the mitochondria. Transport of these alpha-keto acids (and subsequent metabolites) must occur across the mitochondrial inner membrane. We have characterized a specific mitochondrial transport system for branched chain alpha-keto acids which is separate from the second monocarboxylate transporter, the pyruvate carrier. Kinetically, however, these transporters are very similar. Purification of the branched chain alpha-keto acid transporter has resulted in the novel discovery that the mitochondrial aminotransferase, BCAT-m, is a bifunctional protein catalyzing transport of branched chain alpha-keto acids and transamination. Our goal is to determine the mechanism of BCAT-m catalyzed transport and the molecular architecture of the active site. This study will include using covalent modifying reagents to identify amino acid residues that participate in the transport and transaminase activities of BCAT-m. Experiments will be initiated to determine the crystal structure of the branched chain aminotransferases isoenzymes. As part of our third goal of developing a molecular model of mitochondrial monocarboxylate transport, we will clone the cDNA for the pyruvate transporter. This study should provide new information on the diversity of anion transport mechanisms.