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: 3251031
• 负责人: Kelvin J. A. Davies
• 金额: $ 19.59万
• 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: 1985-06-15 至 1993-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3251031
• 关键词: aminoacid analyzer; antioxidants; autoradiography; blood proteins; caseins; catalase; cell free system; complementary DNA; electrofocusing; enzyme mechanism; erythrocytes; fluorimetry; free radicals; gel filtration chromatography; glutathione peroxidase; hemoglobin; high performance liquid chromatography; hydrogen peroxide; hydroxyl group; laboratory rabbit; lipid peroxides; membrane proteins; molecular cloning; peptidases; protein structure; proteolysis; radiotracer; reticulocytes; scintillation counter; serum albumin; singlet oxygen; spectrometry; superoxide dismutase; superoxides

In recent years it has become increasingly apparent that free radicals, and related oxidants, play important roles numerous toxicological processes. The long-term goal of this research is to understand the mechanisms of cellular antioxidant protection against environmental and metabolic toxins. The objective of this application is to test the following hypothesis; "Oxygen radicals and other activated oxygen species, oxidatively denature or fragment hemoglobin, superoxide dismutase, and other red blood cell proteins. Such modified proteins are recognized and rapidly degraded by a red cell proteolytic system which includes a high molecular weight (approximately 700-kDa) ATP- independent, neutral, endo-protease, and several peptidases. By preventing the aggregation of denatured proteins, as well as the potential toxicity of protein fragments, this proteolytic system acts as a line of "Secondary Antioxidant Defense" for the red cell. Secondary Antioxidant functions for proteolytic systems in other cells may be a general biological phenomenon. The specific aims of this proposal are to quantify and describe protein damage and degradation in intact erythrocytes and reticulocytes, the modification of proteins (particularly hemoglobin and superoxide dismutase) by active oxygen species, the degradation of modified proteins in cell-free extracts of red blood cells, and comparative measurements in E. coli. Major specific aims also include, the purification and characterization of the red cell 700-kDa protease, production of polyclonal antibodies against the protease and its subunits, and cloning the protease subunits in E. coli (using a phage expression vector). The approaches and methodology include: proteolysis measures with cell, and purified (labeled) protein modification by spectrophotometry, fluorometry, scintillation counting, HPLC, LC, electrophoresis, and IEF; chromatographic isolation of the 700-kDa protease, generation of antibodies against the protease, and construction of an affinity column; cloning the protease subunits in bacteria and construction of a cDNA library (using a lambda gt11 expression vector). The relationship to health concerns involves: 1) The toxicity of many "rodox active" xenobiotics (environmental agents and drugs); 2) The defenses of cells against oxidative stresses, in health and disease; 3) Adaptive responses to toxins and stress.

近年来，越来越明显的是自由 自由基和相关氧化剂，扮演着重要的角色 毒理学过程。 这项研究的长期目标是了解机制 细胞抗氧化剂保护环境和 代谢毒素。 此应用的目的是测试以下 假设; “氧自由基和其他活化的氧气， 氧化性变性或碎片血红蛋白，超氧化物歧化酶， 和其他红细胞蛋白。 这种修饰的蛋白质是 被红细胞蛋白水解系统识别并迅速降解 其中包括高分子量（约700 kDa）ATP- 独立，中性，内蛋白酶和几种肽酶。 经过 防止变性蛋白的聚集以及 蛋白质碎片的潜在毒性，该蛋白水解系统 充当红细胞的“次级抗氧化剂防御”线。 蛋白水解系统的二级抗氧化功能 细胞可能是一种一般的生物学现象。 该提案的具体目的是量化和描述 完整红细胞的蛋白质损害和降解和降解 网状细胞，蛋白质的修饰（尤其是 血红蛋白和超氧化物歧化酶）由活性氧， 未经细胞的红血提取物中改性蛋白的降解 细胞和大肠杆菌中的比较测量。 主要特定 目的还包括净化和表征 红细胞700-kDa蛋白酶，多克隆抗体的产生 针对蛋白酶及其亚基，并克隆蛋白酶 大肠杆菌中的亚基（使用噬菌体表达载体）。 方法和方法包括：蛋白水解测量 细胞，并通过纯化（标记）蛋白质修饰 分光光度法，荧光测定法，闪烁计数，HPLC，LC， 电泳和IEF； 700 kDA的色谱隔离 蛋白酶，针对蛋白酶的抗体产生，以及 建造亲和力柱；克隆蛋白酶亚基 在细菌和建造cDNA库中（使用lambda gt11 表达矢量）。 与健康问题的关系涉及：1） 许多“ Rodox活性”异种生物（环境药物和药物）； 2）细胞防御氧化应激，健康和 疾病; 3）对毒素和压力的适应性反应。