MITOCHONDRIAL PYRUVATE AND TRICARBOXYLATE TRANSPORTERS

该项目的长期目标是更好地了解： 1)丙酮酸（a） 柠檬酸盐（一种单羧酸盐）和柠檬酸盐（一种三羧酸盐） 穿过线粒体内膜;和2） 调节催化这些过程的转运蛋白 (i.e.,丙酮酸和三羧酸转运系统）通过非 与代谢中间体的共价相互作用， 和疾病状态。 这项建议是一项支持请求， 研究丙酮酸/H+同向转运体和 大鼠肝三羧酸-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/化学水平的结构和功能， 关于生理/病理效应物的调节， 药理学试剂。 具体而言，将进行实验 1）确定所需的最佳条件， 提取、稳定化和功能重建（在 蛋白脂质体）的线粒体丙酮酸和三羧酸 2）将这些载体纯化至均匀， 功能形式; 3）识别氨基酸的类型和数量 在这两种运输工具中， 可能存在于底物结合位点内; 4） 确定推定底物的氨基酸序列 结合结构域;和5）表征 提出的生理/病理调节以及 药物对纯化的 运输机 这些研究既是必要的，也是基础性的 我们对分子/化学机制的理解， 哪些阴离子是通过生物膜运输的， 代谢中间体和药理学的潜在能力 直接调节阴离子转运蛋白功能的试剂 在正常和疾病状态下。

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.