ISOLATION OF ELONGATION ENZYMES & SITE OF SYNTHESIS

这个建议是我们实验工作的延续

This proposal is a continuation of our experimental work concerned with the isolation and purification of each of the enzymatic components of the fatty acid chain elongation system present in the hepatic endoplasmic reticulum. Having recently purified, to apparent homogeneity, two of the components, namely, NADPH-specific trans-2-enoyl CoA reductase and beta- hydroxyacyl CoA dehydrase, focus will turn to isolation of the presumed rate-limiting condensing enzyme and the NAD(P)H- dependent-beta-ketoacyl CoA reductase, the former purified by a series of affinity columns, while the latter utilizing a polyethylene glycol gradient following detergent solubilization. Most intriguing at this time, is the mechanism of action of the beta-ketoacyl CoA reductase since this step requires electron input from cytochrome b5 via NADH cytochrome b5 reductase or NADPH cytochrome P-450 reductase. Antibodies to the component enzymes will be prepared from rabbits, purified by employing Protein A sepharose and utilized to study turnover of the enzyme components in the microsomal membrane and to study the site of biogenesis of each enzyme and its insertion into the smooth endoplasmic reticulum as a "complex" or as separate components. Recent studies have led us to question the current belief of the existence of several elongation pathways in liver microsomes. Therefore, attempts will be made to substantiate or disprove our hypothesis that only one elongation system exists, but funneled into the system are multiple condensing enzymes. The role of phospholipid and other lipids in the modulation of component activities will be investigated. Efforts will continue on the elucidation of the interaction between the acyl CoA substrate and intermediates and the active site of each of the enzymes. Attempts to explore the membrane topography of the elongation system will include the use of various proteolytic enzymes and antibodies to the elongation enzymes. Finally, attention will be focused on those factors which play a role in the regulation of the fatty acid chain elongation system. Such factors will include diet, the use of pharmacologic agents such as, clofibrate and cholestyramine which induce hypolipidemia, potent inhibitors of the elongation system like acetylenic acid derivatives, and other peroxisomal proliferators like DEHP which affect both beta-oxidation and chain elongation. Insights into these proposed aims will provide significant contributions to our fundamental understanding of lipid metabolism.