OXYGEN RADICAL TOXICITY AND RED CELL PROTEIN DEGRADATION

近年来，越来越明显的是，免费 自由基和相关的氧化剂在许多方面起着重要作用 毒理过程。 这项研究的长期目标是了解其机制。 针对环境和环境的细胞抗氧化保护 代谢毒素。 此应用程序的目标是测试以下内容 假设；“氧自由基和其他活性氧物种， 氧化变性或碎片化的血红蛋白，超氧化物歧化酶， 和其他红血球蛋白。这种修饰的蛋白质是 被红细胞蛋白分解系统识别和快速降解 它包括一个高分子量(约700 kDa)的ATP- 独立的、中性的、内切酶和几种多肽酶。通过 防止变性蛋白质的聚集，以及 蛋白质片段的潜在毒性，这种蛋白质分解系统 作为红血球的一道“二级抗氧化剂防线”。 蛋白质分解系统的次生抗氧化剂作用 细胞可能是一种普遍的生物现象。 这项提案的具体目标是量化和描述 完整红细胞中蛋白质的损伤和降解 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)通过活性氧物种， 红血无细胞提取液中修饰蛋白的降解 细胞，以及在大肠杆菌中的比较测量。主要详细说明 目标还包括，提纯和表征 红细胞700 kDa蛋白水解酶，多克隆抗体的产生 抗该酶及其亚基，并克隆该酶 大肠杆菌中的亚基(使用噬菌体表达载体)。 这些途径和方法包括：蛋白质分解措施 细胞，并通过纯化(标记)蛋白质修饰 分光光度法、荧光法、闪烁计数法、高效液相 电泳法和离子交换法；700 kDa的层析分离 蛋白水解酶，产生针对该蛋白水解酶的抗体 亲和层析柱的构建及蛋白酶亚基的克隆 在细菌中的表达和cDNA文库的构建(使用波长gt11 表达载体)。 与健康问题的关系涉及：1) 许多具有“氧化还原活性”的外源生物(环境制剂和药物)； 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。

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.