Sundseth S S, Waxman D J
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1990 Sep 5;265(25):15090-5.
Cholesterol 7 alpha-hydroxylase (P-450 Ch7 alpha) catalyzes the first and rate-limiting step in the hepatic conversion of cholesterol to bile acids. P-450 Ch7 alpha activity in rat liver is regulated at three independent levels: (a) feedback inhibition by bile acids (long term regulation); (b) midterm regulation through the diurnal cycle; (c) short term modulation by hormones and dietary factors. P-450 Ch7 alpha was purified to apparent homogeneity and in active form (turnover number = 10-15 min-1 P-450(-1)) from cholestyramine-fed female rats, and rabbit anti-P-450 Ch7 alpha polyclonal antibodies were then prepared. Liver microsomes were isolated from rats fed normal diet or diet containing the bile acid sequestrant cholestyramine and were then killed at either the apex (midnight) or nadir (noon) of the diurnal rhythm of P-450 Ch7 alpha activity. Direct comparison of microsomal P-450 Ch7 alpha enzyme activity levels with P-450 Ch7 alpha protein (Western blotting) and mRNA levels (Northern and slot blots) revealed that the 2.5-3-fold induction of P-450 Ch7 alpha activity with cholestyramine feeding can be fully accounted for by an increase in P-450 Ch7 alpha protein and mRNA. Turnover numbers of 7-9 nmol of 7 alpha-hydroxycholesterol/min/nmol of microsomal P-450 Ch7 alpha were observed for both induced and uninduced animals. Similarly, the postmidnight decrease in enzyme activity could be generally accounted for by a decrease in P-450 Ch7 alpha protein and mRNA, suggesting that these species have relatively short half-lives. The short term regulation of P-450 Ch7 alpha was examined following treatment with the cholesterol precursor mevalonic acid. A 2.5-fold increase in hepatic microsomal P-450 Ch7 alpha activity occurred within 150 min and was accompanied by a significant elevation of P-450 Ch7 alpha mRNA (up to 3-6-fold increase). These findings establish that hepatic cholesterol 7 alpha-hydroxylase activity is regulated in response to long term, midterm, and short term control factors primarily at a pretranslational level and that this regulation is of greater importance than proposed mechanisms based on allosteric effects of bile acids on P-450 Ch7 alpha protein, changes in cholesterol availability, or reversible phosphorylation of a putative P-450 Ch7 alpha phosphoprotein.
胆固醇7α-羟化酶（P-450 Ch7α）催化肝脏中胆固醇转化为胆汁酸的第一步且是限速步骤。大鼠肝脏中P-450 Ch7α的活性在三个独立水平受到调节：（a）胆汁酸的反馈抑制（长期调节）；（b）通过昼夜节律进行的中期调节；（c）激素和饮食因素的短期调节。从服用消胆胺的雌性大鼠中纯化出了具有明显同质性且呈活性形式的P-450 Ch7α（周转数 = 10 - 15分钟-1 P-450(-1)），随后制备了兔抗P-450 Ch7α多克隆抗体。从喂食正常饮食或含胆汁酸螯合剂消胆胺饮食的大鼠中分离肝脏微粒体，然后在P-450 Ch7α活性昼夜节律的峰值（午夜）或谷值（中午）处死大鼠。将微粒体P-450 Ch7α酶活性水平与P-450 Ch7α蛋白（蛋白质免疫印迹法）和mRNA水平（Northern印迹法和狭缝印迹法）直接比较发现，喂食消胆胺后P-450 Ch7α活性2.5 - 3倍的诱导可完全由P-450 Ch7α蛋白和mRNA的增加来解释。对于诱导和未诱导的动物，均观察到7α-羟基胆固醇的周转数为7 - 9 nmol/分钟/ nmol微粒体P-450 Ch7α。同样，午夜后酶活性的降低通常可由P-450 Ch7α蛋白和mRNA的减少来解释，这表明这些物质的半衰期相对较短。在用胆固醇前体甲羟戊酸处理后，研究了P-450 Ch7α的短期调节。肝脏微粒体P-450 Ch7α活性在150分钟内增加了2.5倍，并伴随着P-450 Ch7α mRNA的显著升高（高达3 - 6倍增加）。这些发现表明，肝脏胆固醇7α-羟化酶活性主要在翻译前水平响应长期、中期和短期控制因素进行调节，并且这种调节比基于胆汁酸对P-450 Ch7α蛋白的变构效应、胆固醇可用性变化或假定的P-450 Ch7α磷蛋白的可逆磷酸化所提出的机制更为重要。
