Ingelman-Sundberg M, Haaparanta T, Rydström J
Biochemistry. 1981 Jul 7;20(14):4100-6. doi: 10.1021/bi00517a024.
The phospholipid specificity of rabbit liver microsomal cytochrome P-450LM2 catalyzed hydroxylation reactions was examined in reconstituted phospholipid vesicles. An apparent linear relationship between the negative charge of the vesicles and the rate of P-450LM2-catalyzed O-dealkylation of p-nitroanisole or 7-ethoxycoumarin was obtained. The membrane charge-mediated increase in hydroxylation activities was found not to be due to (i) an altered lipid/water partition coefficient of the substrate, (ii) a change in the apparent Michaelis constant of P-450LM2 for the substrate, (iii) a different activation energy of the O-demethylation of p-nitroanisole, (iv) different spin states of P-450LM2 or (v) an altered secondary structure of this enzyme as monitored by circular dichroism. However, when the formation of the ferrous carbonyl complex of P-450LM2 was followed under aerobic or anaerobic conditions after the addition of NADPH to the vesicles, an increased negative charge of the membrane was accompanied by an increased reducibility of P-450LM2. A similar linear relationship between the reducibility of cytochrome b5 and the negative charge of the liposomes was also evident in membranes containing NADPH-cytochrome P-450 reductase and cytochrome b5. It is proposed that the interaction of the reductase with P-450LM2 is inefficient in neutral vesicles and thus rate determining for the overall hydroxylation activities.
在重构的磷脂囊泡中检测了兔肝微粒体细胞色素P - 450LM2催化的羟基化反应的磷脂特异性。囊泡的负电荷与P - 450LM2催化的对硝基苯甲醚或7 - 乙氧基香豆素的O - 脱烷基化速率之间呈现出明显的线性关系。发现膜电荷介导的羟基化活性增加并非由于：(i) 底物的脂/水分配系数改变；(ii) P - 450LM2对底物的表观米氏常数变化；(iii) 对硝基苯甲醚O - 去甲基化的活化能不同；(iv) P - 450LM2的不同自旋态；或(v) 通过圆二色性监测的该酶二级结构改变。然而，当向囊泡中加入NADPH后，在有氧或无氧条件下跟踪P - 450LM2的亚铁羰基配合物形成时，膜负电荷增加伴随着P - 450LM2的还原能力增强。在含有NADPH - 细胞色素P - 450还原酶和细胞色素b5的膜中，细胞色素b5的还原能力与脂质体的负电荷之间也呈现出类似的线性关系。有人提出，在中性囊泡中还原酶与P - 450LM2的相互作用效率低下，因此是整体羟基化活性的速率决定因素。
