BRANCHED CHAIN AMINO ACID METABOLISM IN SKELETAL MUSCLE

人们对人体必需物质的分解代谢有相当大的兴趣 支链氨基酸(亮氨酸、异亮氨酸、缬氨酸)，因为 这些代谢物的营养、生化和临床意义。 支链氨基酸的体内浓度被调节为 疾病的严重新陈代谢后果证明了这一点 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基作用。 转氨酶形成支链α-酮酸。我们的 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 对纯化的酶进行了详细的动力学分析。要了解其中的联系 支链氨基酸转氨酶、线粒体转运之间 和体内氮代谢，一种细胞代谢的体外模型 将使用分离的线粒体进行开发，并在未来保持完整 细胞。 转氨基后，支链α-酮酸的氧化 发生在线粒体中。这些α-酮酸的运输(和 后续代谢物)必须跨线粒体内部发生 薄膜。我们已经确定了一种特定的线粒体运输系统 对于与第二个分开的支链α-酮酸 单羧酸盐转运体，丙酮酸载体。然而，从运动学上讲， 这些运输机非常相似。支链的提纯 阿尔法酮酸转运体的新发现 线粒体氨基转移酶，BCAT-m是一种双功能蛋白 催化支链α-酮酸的运输和 转氨酶。我们的目标是确定BCAT-m的机制 催化转运和活性中心的分子结构。 这项研究将包括使用共价修饰试剂来识别 参与转运和转氨酶的氨基酸残基 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。AS 我们开发线粒体分子模型的第三个目标的一部分 单羧酸转运，我们将克隆丙酮酸的cDNA 传送器。这项研究应该提供关于多样性的新信息。 阴离子传输机制的研究。

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.