MITOCHONDRIAL PI (H+) AND PI DICARBOXYLATE TRANSPORTER

膜转运蛋白是生物体所必需的。 的 对于任何输运，溶质输运的机制都不是很清楚 蛋白质和只有非常少的转运蛋白已经被纯化成这样的蛋白质。 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研究 线粒体磷酸盐转运蛋白（PTP）， 转运蛋白（DTP），以及它们与腺嘌呤的关系 核苷酸转位酶（ANT）。 两者以相似的亲和力结合磷酸盐。 PTP最可能与Pi-共运输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 转运蛋白的结合位点，转运的分子机制 一般的蛋白质催化，以及可能的差异/协调 这些转运蛋白在各种组织中的表达。 PTP已经 提纯 DTP尚未确定。 我们计划（PTP）：（a）消除 ANT和来自转运活性PTP的次要低mw蛋白;（B）完成 通过蛋白质生化技术和cDNA技术确定其一级序列 测序（也称为ANT）;这将产生前导序列和预测 它的二级/三级结构在膜;允许我们准备 不同生物体（和器官）PTP cDNA鉴定部分蛋白 基于共同同源性的高功能重要性序列; 将尝试产生体外点突变以产生 其他关键功能信息;大鼠心脏的PTP（和ANT） 和肝脏在SDS凝胶中具有不同的迁移率，原因在于： 它们的结构基因或心脏/肝脏处理的信号蛋白酶 线粒体;（c）鉴定转运位点的氨基酸残基， 在膜的C和M侧，以及在蛋白质区域相互作用 与脂质或其他蛋白质进行详细的动力学研究， 试剂和（单克隆）抗体;我们希望检测膜PsipH 和/或磷酸盐诱导的蛋白质构象变化;（d）鉴定 PTP和最小尺寸肽的寡聚状态能够催化 （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， DTP鉴定、纯化、重组和表征方法 对PTP开发的产品进行了最小程度的修改。 我们将使用cDNA 制备转运蛋白以分离和表征基因组 基因，以帮助我们解释协调/差异表达这些 不同组织中的蛋白质。

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.