Nguyen L B, Shefer S, Salen G, Ness G, Tanaka R D, Packin V, Thomas P, Shore V, Batta A
Department of Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark 07103.
J Biol Chem. 1990 Mar 15;265(8):4541-6.
Cholesterol 7 alpha-hydroxylase, the cytochrome P-450-dependent and rate-controlling enzyme of bile acid synthesis, was purified from rat and human liver microsomes. The purified fractions were assayed in a reconstituted system containing [4-14C]cholesterol, and cholesterol 7 alpha-hydroxylase activities in these fractions increased 500-600-fold relative to whole microsomes. Polyacrylamide gel electrophoresis of rat microsomes followed by immunoblotting with polyclonal rabbit antisera raised against purified cholesterol 7 alpha-hydroxylases revealed two peaks at molecular masses of 47,000 and 49,000 daltons for both rat and human fractions. Increasing amounts of rabbit anti-rat and anti-human antibodies progressively inhibited rat microsomal cholesterol 7 alpha-hydroxylase activity up to 80%. In contrast, monospecific antibodies raised against other purified cytochrome P-450 enzymes (P-450f, P-450g, and P-450j) did not inhibit rat or human cholesterol 7 alpha-hydroxylase activity. Immunoblots of rat microsomes with the rabbit anti-rat cholesterol 7 alpha-hydroxylase antibody demonstrated that the antibody reacted quantitatively with the rat microsomal enzyme. Microsomes from cholesterol-fed rats showed increased cholesterol 7 alpha-hydroxylase mass, whereas treatment with pravastatin, an inhibitor of hydroxy-methylglutaryl-coenzyme A reductase, reduced enzyme mass. Microsomes from starved rats contained slightly less cholesterol 7 alpha-hydroxylase protein than chow-fed control rats. These results indicate a similarity in molecular mass, structure, and antigenicity between rat and human cholesterol 7 alpha-hydroxylases; demonstrate the production of inhibiting anti-cholesterol 7 alpha-hydroxylase antibodies that can be used to measure the change in cholesterol 7 alpha-hydroxylase enzyme mass under various conditions; and emphasize the unique structure of cholesterol 7 alpha-hydroxylase with respect to other cytochrome P-450-dependent hydroxylases.
胆固醇7α-羟化酶是胆汁酸合成过程中依赖细胞色素P-450且起速率控制作用的酶，已从大鼠和人肝脏微粒体中纯化出来。在含有[4-14C]胆固醇的重组系统中对纯化的组分进行了检测，这些组分中的胆固醇7α-羟化酶活性相对于整个微粒体增加了500 - 600倍。用针对纯化的胆固醇7α-羟化酶制备的兔多克隆抗血清对大鼠微粒体进行聚丙烯酰胺凝胶电泳，随后进行免疫印迹分析，结果显示大鼠和人组分在分子量为47,000和49,000道尔顿处均出现两个峰。兔抗大鼠和抗人抗体的量逐渐增加，可使大鼠微粒体胆固醇7α-羟化酶活性逐渐受到抑制，最高可达80%。相比之下，针对其他纯化的细胞色素P-450酶（P-450f、P-450g和P-450j）制备的单特异性抗体不会抑制大鼠或人胆固醇7α-羟化酶活性。用兔抗大鼠胆固醇7α-羟化酶抗体对大鼠微粒体进行免疫印迹分析表明，该抗体与大鼠微粒体酶发生了定量反应。喂食胆固醇的大鼠的微粒体显示胆固醇7α-羟化酶量增加，而用羟甲基戊二酰辅酶A还原酶抑制剂普伐他汀处理则降低了酶量。饥饿大鼠的微粒体中胆固醇7α-羟化酶蛋白含量略低于正常喂食的对照大鼠。这些结果表明大鼠和人胆固醇7α-羟化酶在分子量、结构和抗原性方面具有相似性；证明了可产生抑制性抗胆固醇7α-羟化酶抗体，该抗体可用于测量在各种条件下胆固醇7α-羟化酶量的变化；并强调了胆固醇7α-羟化酶相对于其他依赖细胞色素P-450的羟化酶具有独特的结构。
