OXYGEN RADICAL TOXICITY AND RED CELL PROTEIN DEGRADATION

氧自由基毒性和红细胞蛋白降解

基本信息

批准号：
3447667
负责人：
Kelvin J. A. Davies
金额：
$ 5.47万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-02-01 至 1988-01-31
项目状态：
已结题

项目摘要

It has become apparent in recent years that free radical reactions play an important role in numerous toxicological processes. The long-term goal of this research is to understand the role of free radicals in overall protein turnover, in health and disease. The objective of this application is to test the hypothesis that: "Oxygen radicals damage red cell cytoplasmic proteins, thus increasing the susceptibility of those proteins to degradation by the erythrocyte or reticulocyte proteolytic systems." The specific aims of this proposal are to determine: 1) the structural and functional damage to erythrocyte and reticulocyte cytoplasmic proteins (particularly hemoglobin) induced by various oxygen radicals; 2) the extent to which damage proteins are proteolytically degraded; 3) the functional characteristics of the proteolytic system(s); 4) the effectiveness of cellular free radical defenses. The approach to be employed will involve: 1) protein damage and degradation in intact rabbit-erythrocytes and reticulocytes, as well as cell-free extracts, directly exposed to oxygen radicals; 2) oxygen radical damage to purified hemoglobin, and selected other purified red cell cytoplasmic proteins; 3) degradation of oxygen radical-damaged purified proteins during subsequent incubation with erythrocyte or reticulocyte cell-free extracts. The methodology for protein damage and degradation will include: functional studies, optical spectra, gel electrophoresis, isoelectric focusing, HPLC-gel filtration and DEAE chromatography, amino acid analysis, fluorescamine reactivity, radiolabeling of proteins by reductive methyolation, de novo synthesis of radiolabeled reticulocyte proteins, liquid scintillation of acid-soluble and acid-precipitable radiolabels, antioxidants and radical scavengers, proteolytic inhibitors, oxygen radical generating systems (chemical, enzymatic, and radiolytic). The relationship to health concerns involves: 1) the toxicity of many redox active xenobiotics; 2) the defenses of normal and abnormal cells against oxidative stress; 3) the physiological oxidation and proteolytic degradation of cell proteins.

近年来，自由基反应在在许多毒理学过程中发挥着重要作用。这项研究的长期目标是了解免费的作用自由基影响整体蛋白质周转、健康和疾病。此应用程序的目的是检验以下假设：“氧气自由基会破坏红细胞胞质蛋白，从而增加这些蛋白质对红细胞降解的敏感性或网织红细胞蛋白水解系统。” 该提案的具体目标是确定：1）结构和红细胞和网织红细胞胞质蛋白的功能损伤（特别是血红蛋白）由各种氧自由基诱导； 2）范围损伤蛋白被蛋白水解降解； 3）功能性蛋白水解系统的特征； 4）有效性细胞自由基防御。采用的方法包括：1）蛋白质损伤和完整兔红细胞和网织红细胞的降解，以及无细胞提取物，直接暴露于氧自由基； 2）氧自由基破坏纯化血红蛋白，并选择其他纯化红细胞细胞质蛋白； 3) 氧自由基损伤的降解纯化随后与红细胞或网织红细胞孵育期间的蛋白质无细胞提取物。蛋白质损伤和降解的方法包括：功能研究、光谱、凝胶电泳、等电聚焦、HPLC-凝胶过滤和 DEAE 色谱、氨基酸分析、荧光胺反应性，通过还原对蛋白质进行放射性标记甲基化，放射性标记网织红细胞蛋白的从头合成，酸溶性和酸沉淀性放射性标记的液体闪烁，抗氧化剂和自由基清除剂、蛋白水解抑制剂、氧自由基发生系统（化学、酶促和辐射分解）。与健康问题的关系涉及：1）许多物质的毒性氧化还原活性异生素； 2）正常和异常细胞的防御对抗氧化应激； 3）生理氧化和蛋白水解细胞蛋白质的降解。