MITOCHONDRIAL PI (H+) AND PI DICARBOXYLATE TRANSPORTER

线粒体 PI (H ) 和 PI 二羧酸转运蛋白

• 批准号: 3282978
• 负责人: Hartmut none Wohlrab
• 金额: $ 21.63万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1987-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3282978
• 关键词: acidity /alkalinity; adenine nucleotides; cell membrane; chemical structure function; complementary DNA; dicarboxylate; gel electrophoresis; heart; liver; mitochondria; nuclear magnetic resonance spectroscopy; phosphates; phospholipids; point mutation; radiotracer; thiols

Membrane transport proteins are essential to living organisms. The mechanism of solute transport is not well understood for any transport protein and only very few transport proteins have been purified to such an extent as to make studies of the catalytic mechanism possible. The purpose of this research project is to investigate the molecular mechanism of the mitochondrial phosphate transport protein (PTP), the dicarboxylate transport protein (DTP), as well as their relationship to the adenine nucleotide translocase (ANT). Both bind phosphate with similar affinity. PTP most likely cotransports H+ with Pi- while DTP exchanges divalent anions, including Pi-2. These studies will help us understand phosphate binding sites of transport proteins, molecular mechanisms of transport protein catalysis in general, and possibly the differential/coordinated expression of these transport proteins in various tissues. PTP has been purified. DTP has not been identified. We plan (PTP): (a) to eliminate ANT and minor low mw proteins from the transport-active PTP; (b) to finish determining its primary sequence by protein biochemical techniques and cDNA sequencing (also ANT); this will yield leader sequence(s) and predictions for its secondary/tertiary structure in the membrane; permit us to prepare PTP cDNA from different organisms (and organs) to identify partial protein sequences of high functional importance on the basis of common homologies; attempts will be made to generate in vitro point mutations to generate additional critical functional information; PTP (and ANT) from rat heart and liver have different mobilities in SDS gels due to differences in: their structural genes or signal protease processing by the heart/liver mitochondria; (c) to identify amino acid residues at the transport site(s), at the C and M side of the membrane, and at protein regions interacting with lipids or other proteins with detailed kinetic studies, with chemical reagents, and (monoclonal) antibodies; we expect to detect membrane PsipH and/or phosphate induced protein conformational changes; (d) to identify the oligomeric state of PTP and minimum size peptide able to catalyze transport; (e) to prepare two or three dimensional PTP crystals for structural studies. We will use photoaffinity substrate analogues to identify DTP, purify, reconstitute, and characterize it with methods minimally modified from those developed for PTP. We will use the cDNA's prepared for the transport proteins to isolate and characterize genomic genes to help us explain coordinated/differential expression of these proteins in different tissues.

膜转运蛋白是生物体所必需的。这个 溶质传输的机制对于任何传输都不是很清楚。 蛋白质，只有极少数的运输蛋白被提纯到这样的 从而使催化机理的研究成为可能。目的 本研究项目的主要目的是探讨黄褐斑形成的分子机制。 线粒体磷酸转运蛋白(PTP)，即二羧酸盐 运输蛋白(DTP)及其与腺嘌呤的关系 核苷酸转位酶(ANT)。两者都以相似的亲和力结合磷酸盐。 PTP很可能与PI-共转运H+，而DTP交换二价 阴离子，包括PI-2。这些研究将帮助我们了解磷酸盐 转运蛋白的结合部位、转运的分子机制 一般的蛋白质催化，可能还有差异/配位 这些转运蛋白在各种组织中的表达。PTP已经被 纯净的。尚未确定DTP。我们计划(PTP)：(A)消除 蚂蚁和次要低分子量蛋白质从转运活性的PTP；(B)完成 用蛋白质生化技术和cDNA法测定其一级序列 测序(也是蚂蚁)；这将产生前导序列(S)和预测 对于它在膜中的二级/三级结构；允许我们制备 利用不同生物(器官)中的PTP基因鉴定部分蛋白质 基于公共同源的具有高功能重要性的序列； 将尝试在体外产生点突变，以产生 其他关键功能信息；来自大鼠心脏的PTP(和ANT) 和肝脏在十二烷基硫酸钠凝胶中具有不同的迁移率，这是由于以下方面的不同： 它们的结构基因或信号蛋白酶由心脏/肝脏处理 线粒体；(C)鉴定运输部位的氨基酸残基(S)， 在膜的C和M侧，以及在相互作用的蛋白质区域 对脂质或其他蛋白质进行详细的动力学研究，用化学物质 试剂和(单抗)抗体；我们希望检测细胞膜PsipH 和/或磷酸盐诱导的蛋白质构象变化；(D)鉴定 PTP的低聚态及其催化的最小尺寸多肽 运输；(E)制备二维或三维PTP晶体 结构研究。我们将使用光亲和底物类似物来 识别DTP、提纯、重构，并使用方法对其进行表征 对为PTP开发的那些进行了最小程度的修改。我们将使用cDNAs 为分离和鉴定基因组而准备的运输蛋白 帮助我们解释这些基因协调/差异表达的基因 不同组织中的蛋白质。