ISOLATION OF ELONGATION ENZYMES & SITE OF SYNTHESIS

这个建议是我们实验工作的继续 分离和纯化每一种 脂肪酸链延长系统的酶组分 存在于肝内质网中。 最近刚刚 将两种组分纯化至表观均一性， 即NADPH特异性反式-2-烯酰辅酶A还原酶和β- 羟酰CoA脱氢酶，重点将转向分离的羟酰CoA脱氢酶， 推测限速缩合酶和NAD（P）H- 依赖性β-酮脂酰辅酶A还原酶，前者通过 一系列亲和柱，而后者利用 在洗涤剂溶解之后，使用聚乙二醇梯度。 此时最有趣的是， β-酮脂酰辅酶A还原酶，因为这一步骤需要电子 通过NADH细胞色素b5还原酶从细胞色素b5输入，或 NADPH细胞色素P-450还原酶。 抗体与 组分酶将从兔子中制备，通过 采用蛋白A琼脂糖凝胶，并用于研究周转 微粒体膜中的酶成分， 研究每种酶的生物发生位点及其插入 滑面内质网是一个“复合体”还是一个单独的 件. 最近的研究让我们质疑 认为肝脏中存在几种延伸途径 微粒体 因此，将试图证实 或者反驳我们的假设只有一个伸长系统 存在，但进入系统的是多重冷凝 内切酶 磷脂和其他脂质在 将研究组件活动的调制。 努力 将继续阐明 酰基辅酶A底物和中间体以及每个的活性位点 的酶。 试图探索膜的地形 伸长系统的使用将包括使用各种 蛋白水解酶和延伸酶的抗体。 最后，我们将集中注意力在那些发挥作用的因素。 在调节脂肪酸链延长系统中的作用。 这些因素包括饮食、药物的使用 如氯贝丁酯和考来烯胺 降脂，延长系统的有效抑制剂，如 炔酸衍生物和其它过氧化物酶体增殖剂 如DEHP，其影响β-氧化和链 伸长率 对这些拟议目标的深入了解将提供 对我们基本理解的重大贡献 脂质代谢

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.