The long term goal of this proposal is to gain an understanding of the system responsible for the peroxidative fragmentation of the polyunsaturated fatty acids of endoplasmic reticulum in response to certain drugs, chemicals and adverse conditions. Preliminary results obtained in the last funded year (1988) of this grant (GM26548-08) suggested that rather than a simple autooxidative, radical-driven reaction, lipid peroxidation may, at least in part, be the result of activity of a previously unreported enzyme. Gaining an understanding of this enzyme, the activity of which has been implicated in drug cytotoxicity, cell necrosis, aging and tumorigenesis, may lead to the eventual control of these conditions. Studies planned include the isolation, purification and characterization of this enzyme, and determination of its kinetics and mechanism of action. The initial solubilization and fractionation schemes will be modified to attempt to maximize yields of active enzyme. Attempts will be made to stabilize the enzyme, which is extremely labile. The enzyme will be characterized with its minimum molecular weight (SDS-PAGE), UV/visible spectrum, possible prosthetic group or metal ion content and amino terminal amino acid sequence. The substrate specificity of the enzyme will be determined, both phospholipid class preference and fatty acyl specificity, using a reconstituted system and commercially available pure phospholipids. Antibodies will be raised to the purified rat liver enzyme in either laying hens or rabbits in order to a) localize the enzyme with respect to subcellular compartment, b) determine the tissue locations of the enzyme and c) to enable isolation of cDNA clones expressing lipid peroxidase protein. The clones will be used both to deduce the complete amino acid sequence of the enzyme and to form probes with which to monitor lipid peroxidase transcription activity in response to drug challenge. The latter, plus antibody use, will aid in identification of the mechanisms by which drugs and chemicals act to trigger lipid peroxidation in vivo, i.e., transcription, or protein or mRNA stabilization.
