MECHANISMS OF PROTECTION AGAINST PEROXIDATIVE DAMAGE

我们之前已经报道过，维生素E是通过酶促再生的 大鼠肝微粒体和人肝中还原型谷胱甘肽(GSH) 微粒体。最近，其他几个组织也提供了支持的证据 然而，参与谷胱甘肽依赖的自然酶(S) 对微粒体脂过氧化的保护作用尚未完全实现 特色化的。拟议研究的主要目标是 阐明维生素E依赖的分子机制 还原型谷胱甘肽对大鼠肝脏脂质过氧化的抑制作用 微粒体。我们的中心假设是至少有两个 机制，一个似乎是维生素E依赖，另一个是维生素 不依赖于E，它们负责抑制脂质 绝对必需的大鼠肝微粒体中的过氧化 GSH和/或GSSG的行动。我们提出的一种机制是 负责依赖GSH的α-淀粉样蛋白的酶促再生 氧化过程中α-生育酚(α-TH)从α-生育酚生成 压力。第二种机制涉及一种蛋白质，这种蛋白质被 硫醇：二硫键交换，可独立于(α-TH)起作用 减少脂质过氧化过程中形成的过氧化脂质。我们的 因此，假设是基于这样一个前提，即对 膜脂质过氧化与特定的蛋白质有关，这些蛋白质是 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)描述 参与(x-TH)再生的GSH依赖蛋白的性质 并探讨其相互作用机制。 GSH，(α-TH和α-TH再生蛋白在体内和 在体外；2)检测GSH/GSSG依赖的蛋白参与 对肝脏脂质过氧化的保护；3)研究其作用 除GSH：GSSG外，生理的，硫醇：二硫化物氧化还原对 抑制膜脂过氧化作用；4)检查颞叶 α-TH、蛋白SH、混合蛋白二硫键之间的关系 形成和脂质过氧化。 包括分子探针的实验程序，如抗体和 将为表现GSH依赖的纯化蛋白生成cDNA 对α-生育酚自由基的还原活性也是如此 至于膜GST同工酶(S)可能受 硫醇：二硫键交换。多层脂质体模型系统将被 被用来研究抑制脂质的详细机制 纯化蛋白的过氧化作用。蛋白质印迹分析和RNA印迹分析 将进行分析以及核连续转录分析 确定改变维生素E营养对其表达的影响 与抑制脂质有关的GSH依赖蛋白的研究进展 过氧化。从这项建议中获得的新信息应该会增强我们的 对自由基致生物损伤控制的认识 膜。

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.