OXYGEN RADICAL TOXICITY AND RED CELL PROTEIN DEGRADATION

氧自由基毒性和红细胞蛋白降解

• 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
• 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
• 来源: https://reporter.nih.gov/project-details/3447667
• 关键词: aminoacid analyzer; antioxidants; autoradiography; blood proteins; catalase; cell free system; electrofocusing; erythrocytes; fluorimetry; free radicals; gel filtration chromatography; glutathione peroxidase; hemoglobin; high performance liquid chromatography; hydrogen peroxide; hydroxyl group; lipid peroxides; peptidases; proteolysis; radiotracer; reticulocytes; scintillation counter; spectrometry; superoxide dismutase; superoxides; aminoacid analyzer; antioxidants; autoradiography; blood proteins; catalase; cell free system; electrofocusing; erythrocytes; fluorimetry; free radicals; gel filtration chromatography; glutathione peroxidase; hemoglobin; high performance liquid chromatography; hydrogen peroxide; hydroxyl group; lipid peroxides; peptidases; proteolysis; radiotracer; reticulocytes; scintillation counter; spectrometry; superoxide dismutase; superoxides

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，自由激进反应发挥的很明显 在众多毒理过程中的重要作用。 这项研究的长期目标是了解自由的作用 整体蛋白质周转，健康和疾病的自由基。 该应用的目的是检验以下假设：“氧气 自由基损害红细胞的细胞质蛋白，从而增加 这些蛋白质对红细胞降解的敏感性或 网状细胞蛋白水解系统。” 该提议的具体目的是确定：1​​）结构和 红细胞和网状细胞细胞质蛋白的功能损害 （特别是血红蛋白）由各种氧自由基诱导； 2）程度 损伤蛋白会蛋白水解降解； 3）功能 蛋白水解系统的特征； 4）有效性 细胞自由基防御。 要使用的方法将涉及：1）蛋白质损伤和 完整的兔子肉芽细胞和网状细胞中的降解以及 无细胞提取物，直接暴露于氧自由基； 2）氧自由基 损害纯化的血红蛋白，并选择其他纯化的红细胞 细胞质蛋白； 3）氧气自由基损害的纯化 随后与红细胞或网状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将包括： 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，通过还原的蛋白质放射性标记 甲基化，从头合成辐射标记的网状细胞蛋白， 酸性溶质和酸性放射性标签的液体闪烁， 抗氧化剂和自由基清除剂，蛋白水解抑制剂，氧自由基 生成系统（化学，酶和放射性物质）。 与健康问题的关系涉及：1）许多人的毒性 氧化还原活性异生物学； 2）正常细胞和异常细胞的防御 反对氧化应激； 3）生理氧化和蛋白水解 细胞蛋白的降解。