BRANCHED CHAIN AMINO ACID METABOLISM IN SKELETAL MUSCLE

本研究的目的是确定生物化学。 支链氨基分解代谢的调控机制 酸(BCAA)、亮氨酸、异亮氨酸和缬氨酸。重点将放在 确定隔间和隔间的代谢后果 运输在监管中的作用。中的前两个步骤 这三种必需氨基酸的分解代谢是 转氨基，然后氧化脱羧基 转氨基产物，支链α-酮酸 (BCKA)。线粒体酶催化第二步， 支链的α-酮酸脱氢酶，是第一个 体内支链氨基酸分解代谢的速率控制步骤，以及遗传 这种酶的损伤导致一组BCKA酸血症 统称为枫糖浆尿病。异常 在血浆中支链氨基酸也会出现在一些临床状态中，并且 研究工作一直致力于可能的临床应用 对于BCAA和BCKA。我们已经确定了一个 线粒体中BCKA的线粒体转运系统 从大鼠心脏中分离出来。因此，我们的第一个目标是了解 BCKA在分子水平上的转运。它的动力学模式 BCKA摄取将在大鼠心脏线粒体中确定。 BCKA和丙酮酸的抑制剂和底物专一性 运输者将被比较，运输者的活动将被 定义在代谢支链氨基酸的组织中。我们的目标是发展 线粒体单羧酸盐的动力学模型：H+ 共转运者。实验将被设计用来净化和 随之而来的是磷脂小泡内重组转运蛋白活性 通过对纯化的蛋白质进行分子鉴定。我们的 第二个目标是了解支链氨基酸的代谢。重点将是 支链氨基酸的亚细胞分布 BCKA的转氨酶与线粒体内定位 脱氢酶调节支链氨基酸的分解代谢。运输的作用 线粒体内的pH值将被检测。使用31P核磁共振 光谱学，线粒体光谱的特征将 为测定完整细胞线粒体的pH值提供了依据。 支链氨基酸转氨酶活性将在亚细胞中进行测定 从大鼠组织中提取的组分和相对于 线粒体标志物酶。初步数据表明， 酶是大鼠心脏中唯一的线粒体。线粒体 酶将从大鼠心脏中提纯，抗体将被提高， 它们将被用来定量支链氨基酸转氨酶 大鼠组织中的抗原。最后，使用分离的线粒体，一个输入 支链氨基酸的体外分解代谢将被开发出来，并用于测试我们的 假设。

The objective of this study is to determine the biochemical mechanisms that regulate catabolism of the branched chain amino acids (BCAA), leucine, isoleucine and valine. Emphasis will be on determining the metabolic consequences of compartmentation and the role of transport in regulation. The first two steps in the catabolism of these three indispensable amino acids are transamination followed by oxidative decarboxylation of the transamination products, the branched chain alpha-keto acids (BCKA). The mitochondrial enzyme catalyzing the second step, the branched chain alpha-keto acid dehydrogenase, is the first rate-controlling step in BCAA catabolism in vivo, and genetic impairment of this enzyme causes a group of BCKA acidemias collectively known as Maple Syrup Urine Disease. Abnormalities in plasma BCAA also occur in a number of clinical states, and research effort has been directed towards possible clinical uses for BCAA and BCKA. We have established the identify of a mitochondrial transport system for BCKA in mitochondria isolated from rat heart. Thus, our first goal is to understand BCKA transport at a molecular level. The kinetic pattern for BCKA uptake will be determined in rat heart mitochondria. Inhibitor and substrate specificities of the BCKA and pyruvate transporters will be compared, and transporter activity will be defined in tissues that metabolize BCAA. Our aim is development of a kinetic model for the mitochondrial monocarboxylate:H+ cotransporters. Experiments will be designed to purify and reconstitute transporter activity in phospholipid vesicles followed by molecular characterization of the purified proteins. Our second goal is to understand BCAA metabolism. Emphasis will be placed on how the subcellular distribution of BCAA aminotransferase and intramitochondrial location of BCKA dehydrogenase regulate BCAA catabolism. The role of transport and intramitochondrial pH will be examined. Using 31P NMR spectroscopy, characterization of the mitochondrial spectra will provide a basis for determining mitochondrial pH in intact cells. BCAA aminotransferase activity will be assayed in subcellular fractions from rat tissues and activity quantitated relative to a mitochondrial marker enzyme. Preliminary data indicate this enzyme is solely mitochondrial in rat heart. The mitochondrial enzyme will be purified from rat heart, antibodies will be raised, and they will be used to quantitate BCAA aminotransferase antigen in rat tissues. Finally, using isolated mitochondria, an in vitro for BCAA catabolism will be developed, and used to test our hypotheses.