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.

我们之前曾报道过，维生素 E 是通过酶促再生的 大鼠肝微粒体中的还原型谷胱甘肽 (GSH)，以及后来在人肝脏中的还原型谷胱甘肽 (GSH) 微粒体。最近其他几个团体也提供了证据支持 这个假设；然而，参与 GSH 依赖性的天然酶 微粒体脂质过氧化的保护尚未完全 特点。拟议研究的主要目标是 描述维生素 E 依赖性的分子机制 还原型谷胱甘肽抑制大鼠肝脏脂质过氧化 微粒体。我们的中心假设是至少有两个 机制，一种似乎是维生素 E 依赖性的，另一种是维生素 E-独立的，负责抑制脂质 表现出绝对需求的大鼠肝微粒体中的过氧化 向 GSH 和/或 GSSG 采取行动。我们建议一种机制是 负责α-谷胱甘肽依赖性酶促再生 氧化过程中来自α-生育酚自由基的生育酚（α-TH） 压力。 第二种机制涉及一种被修饰的蛋白质 硫醇：二硫键交换，其功能可能独立于（α-TH 至 减少脂质过氧化过程中形成的脂质氢过氧化物。我们的 因此，假设的前提是防止 膜脂过氧化与特定蛋白质有关 受生理上重要的巯基和二硫化物调节。 拟议研究的具体目标是 1) 表征 参与 (x-TH) 再生的 GSH 依赖性蛋白的性质 在大鼠肝微粒体中，并研究相互作用的机制 GSH（α-TH 和 α-TH 再生蛋白在体内和 体外; 2) 检查GSH/GSSG依赖性蛋白参与 防止肝脏脂质过氧化； 3）研究效果 除了 GSH:GSSG 之外，生理学硫醇：二硫化物氧化还原对的 抑制膜脂过氧化； 4）检查时间 α-TH、蛋白质SH、混合蛋白质二硫键之间的关系 形成和脂质过氧化。 实验程序包括分子探针，例如抗体和 将为表现出 GSH 依赖性的纯化蛋白生成 cDNA 还具有减少 α-生育酚自由基的活性 至于膜 GST 同工酶，可能受以下因素调节 硫醇：二硫键交换。 多层脂质体模型系统将 用于研究脂质抑制机制的细节 纯化蛋白质的过氧化作用。蛋白质印迹分析和RNA印迹 将进行分析以及核连续转录测定 确定改变维生素 E 营养对表达的影响 参与脂质抑制的 GSH 依赖性蛋白 过氧化。从该提案中获得的新信息应该会增强我们的能力 对控制自由基介导的生物损伤的认识 膜。