MECHANISMS OF PROTECTION AGAINST PEROXIDATIVE DAMAGE

防止过氧化损伤的机制

• 批准号: 2749536
• 负责人: C CHANNA REDDY
• 金额: $ 20.08万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2749536
• 关键词: affinity chromatography; antibody; computer assisted sequence analysis; dietary supplements; enzyme activity; enzyme linked immunosorbent assay; enzyme mechanism; glutathione; high performance liquid chromatography; immunoprecipitation; inhibitor /antagonist; laboratory rabbit; laboratory rat; membrane lipids; microsomes; molecular cloning; nuclear runoff assay; nutrition related tag; oxidative stress; peroxidation; polymerase chain reaction; sulfhydryl reagents; sulfides; tocopherols; vitamin E deficiency; western blottings

We have previously reported that vitamin E is enzymatically regenerated by reduced glutathione (GSH) in rat liver microsomes and later in human liver microsomes. Recently several other groups have provided evidence in support of this hypothesis; however, the nature enzyme(s) involved in GSH-dependent protection against microsomal lipid peroxidation have not been fully characterized. The primary objective of the proposed research is to delineate the molecular mechanisms underling the vitamin E-dependent inhibition of lipid peroxidation by reduced glutathione in rat liver microsomes. Our central hypothesis is that there are at least two mechanisms, one appears to be vitamin E-dependent and the other is vitamin E-independent, which are responsible for the inhibition of lipid peroxidation in rat liver microsomes that exhibit an absolute requirement to GSH and/or GSSG for their action. We propose that one mechanism is responsible for the GSH-dependent enzymatic regeneration of alpha- tocopherol (alpha-TH) from the alpha-tocopheroxyl radical during oxidative stress. The second mechanism involves a protein which is modified by thiol:disulfide exchange, that may function independent of (alpha-TH to reduce lipid hydroperoxides that are formed during lipid peroxidation. Our hypothesis therefore, is based on the premise that protection against membrane lipid peroxidation is associated with specific proteins that are regulated by physiologically important sulfhydryls and disulfides. The specific aims of the proposed research are to 1) characterize the nature of the GSH-dependent protein involved in the regeneration of (x-TH in rat liver microsomes, and to investigate the mechanism of interaction of GSH, (alpha-TH, and the alpha-TH regenerating protein both in vivo and in vitro; 2) examine the GSH/GSSG-dependent protein involved in the protection against lipid peroxidation in the liver; 3) study the effects of physiological, thiol:disulfide redox couples in addition to GSH:GSSG in the inhibition of membrane lipid peroxidation; and 4) examine the temporal relationships among alpaha-TH, protein SH, mixed protein disulfide formation and lipid peroxidation. Experimental procedures to include molecular probes such as antibodies and cDNA will be generated for the purified protein exhibiting GSH-dependent activity towards the reduction of the alpha-tocopheroxyl radical as well as for the membrane GST isoenzyme(s) that may be regulated by thiol:disulfide exchange. Multilamellar liposomal model systems will be employed to investigate details of the mechanism of inhibition of lipid peroxidation by the purified proteins. Western blot analysis and RNA blot analysis as well as nuclear run-on transcriptional assays will be performed to determine the effects of altered vitamin E nutrition on the expression of the GSH-dependent proteins involved in the inhibition of lipid peroxidation. New information derived from this proposal should enhance our understanding on the control of free radical-mediated damage to biological membranes.

我们以前曾报道过维生素E通过 大鼠肝微粒体中的谷胱甘肽（GSH）降低，后来在人肝脏中 微粒体。最近其他几个小组提供了支持的证据 这个假设；但是，与GSH依赖性有关的性质酶 防止微粒体脂质过氧化尚未完全 特征。拟议研究的主要目的是 描述维生素E依赖性的分子机制 通过减少大鼠肝脏的谷胱甘肽抑制脂质过氧化 微粒体。我们的中心假设是至少有两个 机理，一种似乎是维生素E依赖性的，另一种是维生素 电子非依赖性，这是负责抑制脂质的 大鼠肝微粒体的过氧化，表现出绝对需求 向GSH和/或GSSG采取行动。我们建议一种机制是 负责α-依赖GSH依赖性酶再生 氧化过程中α-托吡羟基自由基的生育酚（α-th） 压力。 第二种机制涉及一种蛋白质的修饰 硫醇：二硫化物交换，可能独立于（alpha-th至 减少在脂质过氧化过程中形成的脂质氢过氧化物。我们的 因此，假设是基于这样的前提 膜脂质过氧化与特定蛋白有关 由生理上重要的硫丙烯和二硫化物调节。 拟议研究的具体目的是1）表征 与GSH依赖性蛋白的性质有关（x-th的再生） 在大鼠肝微粒体中，并研究相互作用的机理 GSH（alpha-th和alpha-th-th-th recenerating蛋白在体内和 体外; 2）检查参与 防止肝脏中脂质过氧化； 3）研究效果 生理，硫醇：除GSH外，二硫键氧化还原夫妇：GSSG 抑制膜脂质过氧化； 4）检查时间 阿尔帕哈（Alpaha），蛋白质SH，混合蛋白二硫化物之间的关系 形成和脂质过氧化。 实验程序，包括分子探针，例如抗体和 cDNA将用于纯化的蛋白质，该蛋白具有GSH依赖性 还降低α-铁杉基自由基的活性 至于可能受到调节的膜GST同工酶 硫醇：二硫键交换。 多层脂质体模型系统将是 用于研究抑制脂质机制的细节 纯化的蛋白质过氧化。蛋白质印迹分析和RNA印迹 将进行分析以及核跑步转录测定 确定维生素E营养改变对表达的影响 与脂质抑制有关的GSH依赖性蛋白 过氧化。从该提案中得出的新信息应增强我们的 了解控制生物学的自由基介导的损害 膜。