Atsuta Y, Okuda K
J Lipid Res. 1982 Feb;23(2):345-51.
Hepatic mitochondrial cytochrome P-450 has been partially purified from the non-induced rat liver. The purification consisted of solubilization with cholate, polyethylene glycol fractionation, chromatographic separation using omega-amino-n-octyl Sepharose 4B column, and chromatography on hydroxylapatite. The overall purification of the enzyme from the solubilized extract was about 22-fold on the basis of specific content of cytochrome P-450, and 50-fold on the basis of specific activity. The partially purified enzyme was active for both 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol and 5 beta-cholestane-3 alpha, 7 alpha-diol. That the enzyme activities for these substrates were not due to two different enzymes but to the same active site of a single enzyme protein was shown by several pieces of evidence, i.e., behavior to thermal inactivation, pH-dependency of the reaction velocities, experiments with mixed substrates, and behavior towards inhibitors and activators. The lower Km value and the higher Vmax for 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol compared to 5 beta-cholestane-3 alpha, 7 alpha-diol seem to be important factors for the regulation mechanism that keeps the ratio of cholic acid/chenodeoxycholic acid constant in rat bile.
肝线粒体细胞色素P-450已从未诱导的大鼠肝脏中部分纯化出来。纯化过程包括用胆酸盐溶解、聚乙二醇分级分离、使用ω-氨基-n-辛基琼脂糖4B柱进行色谱分离以及在羟基磷灰石上进行色谱分离。基于细胞色素P-450的比含量，从溶解提取物中对该酶的总体纯化倍数约为22倍，基于比活性则为50倍。部分纯化的酶对5β-胆甾烷-3α,7α,12α-三醇和5β-胆甾烷-3α,7α-二醇均有活性。几条证据表明，这些底物的酶活性并非源于两种不同的酶，而是源于单一酶蛋白的同一活性位点，即热失活行为、反应速度的pH依赖性、混合底物实验以及对抑制剂和激活剂的反应。与5β-胆甾烷-3α,7α-二醇相比，5β-胆甾烷-3α,7α,12α-三醇较低的Km值和较高的Vmax值似乎是维持大鼠胆汁中胆酸/鹅去氧胆酸比例恒定的调节机制的重要因素。
