Physiological And Metabolic Magnetic Resonance Studies

生理和代谢磁共振研究

基本信息

批准号：
8336565
负责人：
Robert E London
金额：
$ 26.15万
依托单位：
NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8336565
关键词：
ATP Synthesis Pathway Acetylcholine Active Sites Anions Bicarbonates Binding Binding Proteins Calcium Carbonates Cell Extracts Cells Characteristics Chemicals Chemistry Choline Cholinergic Antagonists Co-Immunoprecipitations Competitive Binding Complex Consumption Copper Coupled Cuprozinc Superoxide Dismutase Defect Development Disease Dissociation Elements Ensure Environment Enzymes Exposure to Failure Feedback Flagella Genes Genetic Germ Cells Glucose Glycolysis Goals Health Histidine Homeostasis Hydrogen Peroxide Intercept Investigation Ions Isoenzymes Kinetics Label Lactate Dehydrogenase Latrodectus mactans Ligand Binding Ligands Location Magnetic Resonance Male Infertility Marines Mass Spectrum Analysis Mediating Metabolic Metabolic Diseases Metabolism Methodology Methods Molecular Analysis Mutate NADH Nuclear Magnetic Resonance Organ Oxidants Physiological Physiological Processes Positioning Attribute Predisposition Primary Lateral Sclerosis Process Proteins Pyruvate Reaction Reactive Oxygen Species Research Residual state Role Series Signal Transduction Sodium Spiders Sulfhydryl Compounds Superoxide Dismutase Superoxides Suspension substance Suspensions Synapses System Testing Time Toxin absorption analog base carbonate dehydratase cell injury cell motility cellular targeting cofactor copper zinc superoxide dismutase design environmental agent environmental chemical inhibitor/antagonist insight knockout gene lactate dehydrogenase A neurotransmitter metabolism oxidation protective effect response sperm cell sperm function

项目摘要

This project investigates how chemical toxins or physical factors alter metabolic processes. NMR methods provide a unique approach for the investigation of metabolic and physiological processes in intact systems, perfused organs, cell suspensions, as well as by examination of cell extracts. The main studies performed as part of this research effort during the past year are summarized below: Project 1. We previously has been demonstrated that disruption of the germ cell-specific lactate dehydrogenase C gene (Ldhc) results in male infertility due to defects in sperm function that include a rapid decline in sperm ATP levels, a decrease in progressive motility, and a failure to develop hyperactivated motility. We hypothesized that lack of LDHC disrupts glycolysis by feedback inhibition, either by causing a defect in renewal of the NAD+ cofactor essential for activity of GAPDHS, or an accumulation of pyruvate. To test these hypotheses, nuclear magnetic resonance (NMR) analysis was used to follow the utilization of labeled substrates in real time. We found that in sperm lacking LDHC, glucose consumption was disrupted, but the NAD/NADH ratio and pyruvate levels were unchanged, and pyruvate was rapidly metabolized to lactate. Moreover, the metabolic disorder induced by treatment with the LDH inhibitor sodium oxamate was different from that caused by lack of LDHC. This supported a previous proposal that lactate dehydrogenase A, an LDH isozyme present in the principal piece of the flagellum, is responsible for the residual LDH activity in sperm lacking LDHC. By co-immunoprecipitation coupled with mass spectrometry we have identified 27 proteins associated with LDHC. A majority of these proteins are implicated in ATP synthesis, utilization, transport and/or sequestration. On the basis of these results, it appears that LDHC is part of a complex involved in ATP homeostasis that is disrupted in spermatozoa lacking this isozyme. Project 2. Copper-zinc superoxide dismutase (SOD) protects the cell as a consequence of its role in the dismutation of superoxide. However, in the presence of hydrogen peroxide it can also generate a powerful oxidant, the carbonate anion radical, which can in turn oxidize other cellular targets. The mechanism of carbonate radical formation by SOD has been a subject of intense debate. Recent kinetic studies have been designed to provide insight into this process, focusing on the possible role of peroxycarbonate as a substrate for reduced SOD. Based on recent theoretical analyses of SOD, it appears that reduction of the active site Cu(II) to Cu(I) by hydrogen peroxide Is accompanied by release of the bridging histidine ligand to the copper. This may allow larger ions, such as peroxycarbonate, to bind to the Cu(I) enzyme, and the subsequent chemistry might result in formation of the carbonate radical. We have also continued our studies using NMR to evaluate the role of carbonic anhydrase in the metabolism of peroxymonocarbonate. Project 3. We previously used NMR to characterize ligand interactions with a putative acetylcholine binding protein (AChBP) derived from the marine polychaete Capitalla teleta. We have recently begun a related project to investigate ligand binding with an AChBP derived from the black widow spider. The NMR approach that we have followed utilizes competitive binding interactions to allow the determination of dissociation constants much lower than would be feasible using just the ligand and binding protein. These studies begin with a weak binding ligand, such as choline, and subsequently evaluate ligands with decreasing KD values. The spider toxin contains an additional 35 residue extension at the C-terminus, the function of which is unknown.

该项目研究化学毒素或物理因素如何改变代谢过程。 NMR方法为研究完整系统，灌注器官，细胞悬浮液以及通过检查细胞提取物的代谢和生理过程提供了独特的方法。在过去一年中，作为这项研究工作的一部分进行的主要研究总结了：项目1。以前已经证明，由于精子功能的缺陷，包括精子功能的缺陷，促进性ATP水平的迅速下降，进行性运动性的降低以及未能发展多激活的运动能力，导致生殖细胞特异性乳酸脱氢酶C基因（LDHC）的破坏导致男性不育症。我们假设缺乏LDHC通过反馈抑制会破坏糖酵解，这要么是通过续订NAD+辅助因子的缺陷而对GAPDHS活性或丙酮酸的积累所必需的。为了检验这些假设，使用核磁共振（NMR）分析来实时遵循标记的底物的利用。我们发现，在缺乏LDHC的精子中，葡萄糖消耗受到破坏，但是NAD/NADH的比率和丙酮酸水平不变，丙酮酸迅速代谢为乳酸。此外，用LDH抑制剂钠诱导的代谢障碍与缺乏LDHC引起的代谢障碍不同。这支持了先前的提议，即鞭毛脱氢酶A，即鞭毛主要部分中存在的LDH同工酶，负责缺乏LDHC的精子中残留的LDH活性。通过共免疫沉淀，加上质谱法，我们确定了与LDHC相关的27种蛋白质。这些蛋白质中的大多数与ATP合成，利用，运输和/或隔离有关。根据这些结果，LDHC似乎是参与ATP稳态的复合体的一部分，该复合体在缺乏同工酶的精子中被破坏。项目2。铜 - 锌超氧化物歧化酶（SOD）由于其在超氧化物损失中的作用而保护该细胞。但是，在存在过氧化氢的情况下，它也可以产生强大的氧化剂，即碳酸盐阴离子自由基，进而可以氧化其他细胞靶标。 SOD碳酸盐自由基形成的机制一直是激烈争论的主题。最近的动力学研究旨在提供对这一过程的见解，重点是过氧碳酸盐作为减少SOD的底物的可能作用。基于最近对SOD的理论分析，看来通过过氧化氢将活性位点Cu（II）还原为Cu（I），伴随着将桥接的组氨酸配体释放到铜中。这可能会使较大的离子（例如过氧碳酸盐）与Cu（I）酶结合，随后的化学可能导致碳酸盐自由基的形成。我们还使用NMR继续进行研究，以评估碳酸酐酶在过氧甲酸盐代谢中的作用。项目3。我们先前使用NMR来表征与推定的乙酰胆碱结合蛋白（ACHBP）的配体相互作用，该蛋白（ACHBP）衍生自海洋多chaete Capitalla Teleta。我们最近开始了一个相关的项目，以研究与黑寡妇蜘蛛的ACHBP结合。我们遵循的NMR方法利用竞争性结合相互作用，允许确定分离常数远低于仅使用配体和结合蛋白的可行性。这些研究始于弱结合配体（例如胆碱），然后以降低KD值评估配体。蜘蛛毒素在C末端还包含35个残基伸展，其功能尚不清楚。