ELECTRON TRIBUTARIES OF THE RESPIRATORY CHAIN

呼吸链的电子支流

• 批准号: 3272613
• 负责人: YOUSSEF HATEFI
• 金额: $ 22.4万
• 依托单位: SCRIPPS CLINIC AND RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-08-01 至 1991-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3272613
• 关键词: NAD(H) phosphate; NAD(P) transhydrogenase; affinity chromatography; antigens; bioenergetics; chemical group; complementary DNA; conformation; cow; electron transport; enzyme inhibitors; enzyme linked immunosorbent assay; enzyme mechanism; enzyme reconstitution; enzyme structure; enzyme substrate; fluorescence spectrometry; heart; laboratory mouse; laboratory rabbit; laboratory rat; membrane permeability; mitochondria; mitochondrial membrane; monoclonal antibody; nicotinamide adenine dinucleotide; oxidation; phosphatidylcholines; phospholipids; protein sequence; protonation; ubiquinone

The objective is to study the structure and mechanism of action of two membrane-bound enzymes of mitochondria: the energy-linked nicotinamide nucleotide transhydrogenase (TH), and the phospholipid-requiring D(-)Beta-hydroxybutyrate dehydrogenase (BDH). TH is structurally the simplest of the mitochondrial energy-transducing enzymes. It is a single polypeptide which occurs in the membrane and isolated state apparently as a homodimer. Our goal is to understand the mechanism of energy transduction by TH. Proposed studies include: (i) isolation and sequence analysis of active site peptides labeled with (a) [3H]p-fluorosulfonylbenzoyl-5'-adenosine (FSBA) at the NAD(H) site, (b) FSBA at the NADP(H) site, and (c) [14C]DCCD at the NAD(H) site; (ii) effects of substrates (particularly NADP and NADPH) and certain inhibitors on TH conformation; (iii) test of alternating site catalysis as a possible explanation of the apparent half-site reactivity of TH; (iv) production of antibodies to various TH fragments and of monoclonals to different TH epitopes for study of enzyme topography in the mitochondrial inner membrane and possible identification of segments concerned with scalar catalysis and proton translocation; (v) deduce the amino acid sequence of TH from the corresponding cDNA sequence. BDH requires lecithin as cofactor; hence it is a model enzyme for study of protein-lipid interaction. Proposed studies include: (i) isolationa nd sequence analysis of active site peptides labeled with (a) arylazido-[3H]Beta-alanyl NAD (N3-NAD), (b) [14C]N-ethylmaleimide, and (c) [14C]DCCD; (ii) labeling of BDH with arylazidophospholipids, isolation and sequence analysis of labeled peptides; (iii) study of role of essential residues (arginyl, thiol, carboxyl) in catalysis. BDH activity is low to negligible in various hepatoma mitochondria. We also plan to investigate whether this is due to low enzyme level (by ELISA immunoassays) or altered phospholipids of hepatoma mitochondria, and to study how these mitochondria cope with virtual lack of BDH function (do they not produce acetoacetate, do they unlike normal liver mitochondria contain succinyl-CoA:acetoacetate CoA-transferase, or are able to metabolize acetoacetate by an alternate route?).

目的是研究两个的作用结构和机理 线粒体的膜结合酶：能量连接的烟酰胺 核苷酸经氢化酶（Th）和重磷脂的重新测量 D（ - ）β-羟基丁酸脱氢酶（BDH）。 在结构上是 线粒体能量传授酶的最简单。 这是一个 多肽发生在膜中，孤立状态显然是作为一个 同二聚体。 我们的目标是了解能量转移的机制 由Th。 拟议的研究包括：（i）隔离和序列分析 带有（a）标记的活性位点肽 [3H] NAD（H）站点的P-Fluorosulfonylbenzoyl-5'-腺苷（FSBA），（b） NADP（H）站点的FSBA和（C）[14C] DCCD在NAD（H）站点； （ii） 底物（尤其是NADP和NADPH）和某些抑制剂的影响 在构象上； （iii）可能的交替催化测试 解释Th的明显半位点反应性； （iv）生产 对各种片段和单克隆的抗体 用于研究线粒体内膜酶地形的表位 以及可能识别与标量催化和 质子易位； （v）从 相应的cDNA序列。 BDH需要卵磷脂作为辅助因子；因此，这是一种用于研究的模型酶 蛋白 - 脂质相互作用。 拟议的研究包括：（i）隔离 用（a）标记的活性位点肽的序列分析 芳基西多 - [3H]β-丙烷NAD（N3-NAD），（b）[14C] N-乙基甲酰亚胺和（C） [14C] DCCD; （ii）用芳基氮磷脂，分离和 标记肽的序列分析； （iii）研究基本作用 残基（精氨酸，硫醇，羧基）在催化中。 BDH活性低到 在各种肝癌线粒体中都可以忽略不计。 我们还计划调查 这是由于低酶水平（通过ELISA免疫测定）还是改变 线粒体的肝癌的磷脂，并研究这些线粒体如何 应付虚拟缺乏BDH功能（它们不会产生乙酸乙酸盐， 它们是否与正常肝线粒体含有琥珀酸酯：乙酸 COA-转移酶，或能够通过替代品代谢乙酸 路线？）。