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: 3251033
• 负责人: Kelvin J. A. Davies
• 金额: $ 16.55万
• 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/3251033
• 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， 自由基和相关的氧化剂在许多方面都起着重要的作用， 毒理学过程 这项研究的长期目标是了解 细胞抗氧化保护， 代谢毒素 本应用程序的目的是测试以下内容 假设;“氧自由基和其他活性氧物质， 氧化变性或片段血红蛋白，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 其包括高分子量（约700-kDa）ATP- 独立的中性内切蛋白酶和几种肽酶。 通过 防止变性蛋白质的聚集，以及 蛋白质片段的潜在毒性，这种蛋白水解系统 作为红细胞的“二级抗氧化防御”。 其他蛋白水解系统的二级抗氧化功能 细胞可能是一种普遍的生物现象。 本提案的具体目标是量化和描述 完整红细胞中的蛋白质损伤和降解， 网织红细胞，蛋白质的修饰（特别是 血红蛋白和超氧化物歧化酶）的活性氧， 红血无细胞提取物中修饰蛋白质的降解 细胞，并在E.杆菌 主要具体 目的还包括，纯化和表征的 红细胞700-kDa蛋白酶，多克隆抗体的生产 针对蛋白酶及其亚基，并克隆蛋白酶 亚基E. coli（使用噬菌体表达载体）。 方法和方法包括：蛋白水解措施， 细胞和纯化的（标记的）蛋白质修饰， 分光光度法、荧光法、闪烁计数法、HPLC、LC、 电泳和IEF;色谱分离700-kDa 蛋白酶，产生针对蛋白酶的抗体，和 构建亲和柱;克隆蛋白酶亚基 并构建cDNA文库（使用λ> 11 表达载体）。 与健康问题的关系包括：1） 许多具有“氧化还原活性”的外源性物质（环境剂和药物）; 2)细胞对氧化应激的防御，在健康和 疾病; 3）对毒素和压力的适应性反应。