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.
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