Halvorson M, Greenway D, Eberhart D, Fitzgerald K, Parkinson A
Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City 66103.
Arch Biochem Biophys. 1990 Feb 15;277(1):166-80. doi: 10.1016/0003-9861(90)90566-h.
Cytochrome P450p (IIIA1) has been purified from rat liver microsomes by several investigators, but in all cases the purified protein, in contrast to other P450 enzymes, has not been catalytically active when reconstituted with NADPH-cytochrome P450 reductase and dilauroylphosphatidylcholine. We now report the successful reconstitution of testosterone oxidation by cytochrome P450p, which was purified from liver microsomes from troleandomycin-treated rats. The rate of testosterone oxidation was greatest when purified cytochrome P450p (50 pmol/ml) was reconstituted with a fivefold molar excess of NADPH-cytochrome P450 reductase, an equimolar amount of cytochrome b5, 200 micrograms/ml of a chloroform/methanol extract of microsomal lipid (which could not be substituted with dilauroylphosphatidylcholine), and the nonionic detergent, Emulgen 911 (50 micrograms/ml). Testosterone oxidation by cytochrome P450p was optimal at 200 mM potassium phosphate, pH 7.25. In addition to their final concentration, the order of addition of these components was found to influence the catalytic activity of cytochrome P450p. Under these experimental conditions, purified cytochrome P450p converted testosterone to four major and four minor metabolites at an overall rate of 18 nmol/nmol P450p/min (which is comparable to the rate of testosterone oxidation catalyzed by other purified forms of rat liver cytochrome P450). The four major metabolites were 6 beta-hydroxytestosterone (51%), 2 beta-hydroxytestosterone (18%), 15 beta-hydroxytestosterone (11%) and 6-dehydrotestosterone (10%). The four minor metabolites were 18-hydroxytestosterone (3%), 1 beta-hydroxytestosterone (3%), 16 beta-hydroxytestosterone (2%), and androstenedione (2%). With the exception of 16 beta-hydroxytestosterone and androstenedione, the conversion of testosterone to each of these metabolites was inhibited greater than 85% when liver microsomes from various sources were incubated with rabbit polyclonal antibody against cytochrome P450p. This antibody, which recognized two electrophoretically distinct proteins in liver microsomes from troleandomycin-treated rats, did not inhibit testosterone oxidation by cytochromes P450a, P450b, P450h, or P450m. The catalytic turnover of microsomal cytochrome P450p was estimated from the increase in testosterone oxidation and the apparent increase in cytochrome P450 concentration following treatment of liver microsomes from troleandomycin- or erythromycin-induced rats with potassium ferricyanide (which dissociates the cytochrome P450p-inducer complex). Based on this estimate, the catalytic turnover values for purified, reconstituted cytochrome P450p were 4.2 to 4.6 times greater than the rate catalyzed by microsomal cytochrome P450p.
几位研究人员已从大鼠肝脏微粒体中纯化出细胞色素P450p（IIIA1），但在所有情况下，与其他P450酶相比，纯化后的蛋白质在与NADPH - 细胞色素P450还原酶和二月桂酰磷脂酰胆碱重构时均无催化活性。我们现在报告从经曲古抑菌素处理的大鼠肝脏微粒体中纯化的细胞色素P450p成功实现了睾酮氧化的重构。当纯化的细胞色素P450p（50 pmol/ml）与五倍摩尔过量的NADPH - 细胞色素P450还原酶、等摩尔量的细胞色素b5、200微克/毫升的微粒体脂质氯仿/甲醇提取物（不能用二月桂酰磷脂酰胆碱替代）以及非离子去污剂Emulgen 911（50微克/毫升）重构时，睾酮氧化速率最大。细胞色素P450p催化的睾酮氧化在200 mM磷酸钾、pH 7.25时最为适宜。除了它们的终浓度外，还发现这些成分的添加顺序会影响细胞色素P450p的催化活性。在这些实验条件下，纯化的细胞色素P450p以18 nmol/nmol P450p/分钟的总速率将睾酮转化为四种主要代谢物和四种次要代谢物（这与其他纯化形式的大鼠肝脏细胞色素P450催化的睾酮氧化速率相当）。四种主要代谢物为6β - 羟基睾酮（51%）、2β - 羟基睾酮（18%）、15β - 羟基睾酮（11%）和6 - 脱氢睾酮（10%）。四种次要代谢物为18 - 羟基睾酮（3%）、1β - 羟基睾酮（3%）、16β - 羟基睾酮（2%）和雄烯二酮（2%）。除16β - 羟基睾酮和雄烯二酮外，当将来自各种来源的肝脏微粒体与抗细胞色素P450p的兔多克隆抗体一起孵育时，睾酮向这些代谢物中每一种的转化均受到大于85%的抑制。该抗体识别经曲古抑菌素处理的大鼠肝脏微粒体中两种电泳上不同的蛋白质，不抑制细胞色素P450a、P450b、P450h或P450m催化的睾酮氧化。通过用铁氰化钾（它能解离细胞色素P450p - 诱导剂复合物）处理经曲古抑菌素或红霉素诱导的大鼠的肝脏微粒体后，根据睾酮氧化的增加和细胞色素P450浓度的明显增加来估计微粒体细胞色素P450p的催化周转率。基于此估计，纯化的、重构的细胞色素P450p的催化周转率值比微粒体细胞色素P450p催化的速率高4.2至4.6倍。
