MITOCHONDRIAL PYRUVATE AND TRICARBOXYLATE TRANSPORTERS

该项目的长期目标是更好地了解： 1)丙酮酸(A)的分子机制(S 单羧酸盐)和柠檬酸盐(三羧酸盐)被运输 穿过线粒体内膜；和2)线粒体的性质 对催化这些过程的运输蛋白的调节 (即丙酮酸和三羧酸盐运输系统) 两者与代谢中间产物的共价相互作用 和疾病状态。该提案代表对支持的请求 5年来研究丙酮酸/H+转运体和 大鼠肝脏三羧酸盐-H+/二羧酸逆向转运蛋白 线粒体。这些系统将在以下方面进行研究 分子/化学水平上的结构和功能，以及 尊重生理/病理效应器的调节和 药理制剂。具体来说，将进行实验 1)确定满足以下条件的最佳条件： 提取、稳定和功能重建(in 线粒体丙酮酸和三羧酸盐的蛋白质脂质体 转运蛋白；2)将这些载体完全提纯为均一 功能形式；3)确定氨基酸的类型和数量 在两个传送器中，这两个传送器对于运输和 可能驻留在底物结合部位内；4) 确定推测底物的氨基酸序列 结合结构域；以及5)表征了 建议的生理/病理规则以及 药理药物对纯化后细胞功能的影响 传送者。这些研究既是必要的，也是基础的。 对我们对分子/化学机制的理解(S) 哪些阴离子是通过生物膜运输的，以及 代谢中间体的潜在能力与药理作用 直接调节阴离子转运体功能的药物 在正常和患病状态下。

The long range objectives of this project are to better understand: 1) the molecular mechanism(s) by which pyruvate (a monocarboxylate) and citrate (a tricarboxylate) are transported across the mitochondrial inner membrane; and 2) the nature of the regulation of the transport proteins catalyzing these processes (i.e., the pyruvate and tricarboxylate transport systems) by non- covalent interaction with metabolic intermediates in both normal and disease states. This proposal represents a request for support for 5 years to study the pyruvate/H+ symporter and the tricarboxylate-H+/dicarboxylate antiporter of rate liver mitochondria. These systems will be studied with respect to structure and function at the molecular/chemical levels, and with respect to regulation by physiological/pathological effectors and pharmacological agents. Specifically, experiments will be carried out to: 1) Determine the optimal conditions required for the extraction, stabilization, and functional reconstitution (in proteoliposomes) of the mitochondrial pyruvate and tricarboxylate transporters; 2) Purify these carriers to homogeneity in fully functional form; 3) Identify the type and number of amino acids within the two transporters that are essential for transport and are likely to reside within the substrate binding sites; 4) Determine the amino acid sequences of the putative substrate binding domains; and 5) Characterize the direct effect of proposed physiological/pathological regulations as well as pharmacological agents on the functioning of the purified transporters. These studies are both necessary and fundamental to our understanding of the molecular/chemical mechanism(s) by which anions are transported across biological membrane, and the potential ability of metabolic intermediates and pharmacological agents to directly regulate the functioning of anion transporters in normal and diseased states.