Iwahashi K, Tsubaki M, Miyatake A, Miura S, Hosokawa K, Ichikawa Y
Department of Biochemistry, Kagawa Medical School, Japan.
J Steroid Biochem Mol Biol. 1991 Jun;38(6):727-31. doi: 10.1016/0960-0760(91)90085-j.
Cholesterol side-chain cleavage activities of cytochrome P-450ssc purified from bovine adrenocortical mitochondria were measured for various substrates, including cholesterol, 20[S]-hydroxycholesterol, 22[R]-hydroxycholesterol and 20[R], 22[R]-dihydroxycholesterol, in the reconstituted enzyme system at various Tween20 concentrations. The side-chain cleavage activity for cholesterol showed more than 10-fold enhancement upon addition of 0.1% Tween20, compared with that without the detergent. Addition of Tween20 did not cause any enhancement of the side-chain cleavage activities for 20[S]-hydroxycholesterol and 22[R]-hydroxycholesterol; rather, it resulted in an inhibition of the activities. The side-chain cleavage activity for 20[R],22[R]-dihydroxycholesterol showed a very high value even without the detergent. As the stimulatory effect of Tween20 was only specific for cholesterol, Tween20 seemed to enhance the rate of access of cholesterol to cytochrome P-450scc. These results are consistent with the suggestion that a transfer of substrate, cholesterol, in mitochondrial inner membrane, to the substrate-binding site of cytochrome P-450scc is the rate-limiting step in the cholesterol side-chain cleavage reaction.
在重构酶系统中，于不同吐温20浓度下，测定了从牛肾上腺皮质线粒体中纯化的细胞色素P - 450ssc对多种底物（包括胆固醇、20[S]-羟基胆固醇、22[R]-羟基胆固醇和20[R],22[R]-二羟基胆固醇）的胆固醇侧链裂解活性。与未添加去污剂的情况相比，添加0.1%吐温20后，胆固醇的侧链裂解活性增强了10倍以上。吐温20的添加并未使20[S]-羟基胆固醇和22[R]-羟基胆固醇的侧链裂解活性增强；相反，它导致了这些活性的抑制。即使没有去污剂，20[R],22[R]-二羟基胆固醇的侧链裂解活性也显示出非常高的值。由于吐温20的刺激作用仅对胆固醇具有特异性，吐温20似乎提高了胆固醇进入细胞色素P - 450scc的速率。这些结果与以下观点一致，即线粒体内膜中的底物胆固醇向细胞色素P - 450scc底物结合位点的转移是胆固醇侧链裂解反应中的限速步骤。
