ISOLATION OF ELONGATION ENZYMES & SITE OF SYNTHESIS

这项建议是我们实验工作的延续 与分离和提纯每一种 脂肪酸链延长系统的酶组分 存在于肝脏内质网中。最近有过一次 将其中两种成分提纯，达到明显的同质性， 即NADPH特异性的反式-2-烯醇基辅酶A还原酶和β-羟基辅酶A。 羟酰辅酶A脱水酶，焦点将转向分离 推测的限速缩合酶和NAD(P)H- 依赖的β-酮酰辅酶A还原酶，前者经纯化 一系列亲和柱，而后者使用 洗涤剂增溶后的聚乙二醇梯度。 在这个时候最耐人寻味的是 β-酮酰辅酶A还原酶，因为这一步需要电子 细胞色素b5通过NADH细胞色素b5还原酶或 NADPH细胞色素P-450还原酶。针对该病毒的抗体 成分酶将从兔子中制备，通过以下方法纯化 蛋白A琼脂糖凝胶用于研究蛋白质的周转 微粒体膜中的酶组分和TO 研究每种酶的生物发生部位及其插入 光滑的内质网是一个“复合体”，也可以是独立的 组件。最近的研究让我们对当前的 相信肝脏中存在几种伸长途径 微粒体。因此，将努力证明 或者反驳我们的假设，即只有一种伸长系统 存在，但流入系统的是多个冷凝 酵素。磷脂和其他脂类在血管紧张素转换酶中的作用 组分活动的调节将被调查。努力 将继续阐明 酰基辅酶A底物和中间体及其活性中心 酶的作用。膜形貌的探索尝试 延伸率系统的使用将包括各种 蛋白水解酶和对伸长酶的抗体。 最后，我们将把注意力集中在这些因素上。 在调节脂肪酸链延长系统中的作用。 这些因素将包括饮食、药剂的使用 例如氯贝特和氯苯乙胺，它们可以诱导 低脂血症，延伸系统的有效抑制物，如 乙炔酸衍生物和其他过氧化体增殖剂 像DEHP一样，既影响β-氧化又影响链 伸长率。对这些拟议目标的洞察将提供 对我们从根本上理解 脂类代谢。

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.