OXYGEN RADICAL TOXICITY AND RED CELL PROTEIN DEGRADATION

近年来，自由基反应在其中起着重要作用， 在许多毒理学过程中发挥重要作用。 这项研究的长期目标是了解免费的作用， 自由基在整体蛋白质周转，在健康和疾病。 本申请的目的是检验以下假设：“氧气 自由基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。" 本建议的具体目标是确定：1）结构和 红细胞和网织红细胞胞质蛋白功能损伤 （特别是血红蛋白）诱导的各种氧自由基; 2）程度 蛋白质被蛋白水解降解; 3）功能性 蛋白水解系统的特性; 4） 细胞自由基防御系统 所采用的方法将涉及：1）蛋白质损伤， 在完整家兔红细胞和网织红细胞中降解，以及 无细胞提取物，直接暴露于氧自由基; 2）氧自由基 对纯化血红蛋白和选定的其他纯化红细胞的损害 细胞质蛋白质; 3）降解氧自由基损伤的纯化 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，通过还原剂对蛋白质进行放射性标记 羟甲基化，放射性标记的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 生成系统（化学、酶和辐射分解）。 与健康问题的关系涉及：1）许多药物的毒性 氧化还原活性异生物质; 2）正常和异常细胞的防御 抗氧化应激; 3）生理氧化和蛋白水解 细胞蛋白质的降解。

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.