Ingelman-Sundberg M, Blanck J, Smettan G, Ruckpaul K
Eur J Biochem. 1983 Jul 15;134(1):157-62. doi: 10.1111/j.1432-1033.1983.tb07546.x.
The kinetics of the reduction of cytochrome P-450 LM2 mediated by NADPH-cytochrome P-450 reductase in reconstituted phospholipid vesicles was examined. An inefficient reduction of the hemoprotein in phosphatidylcholine vesicles was observed. However, by introducing negatively charged phospholipids into the membrane, the rate of reduction increased in a concomitant manner to the resulting net negative charge of the vesicles. In the presence of benzphetamine, the extent of cytochrome P-450 LM2 reduced 1 s after the addition of NADPH to the system was a linear function of the electrophoretic mobilities of the vesicles used. A similar relationship between the net negative charge of the vesicles, as measured electrophoretically, and the reduction rate was also attained in the absence of substrate. The enhanced reduction was mainly reflected in an altered phase distribution of the reduction; the extent of fast phase reduction in the absence or in the presence of added substrate was dependent upon the electrophoretic mobilities of the vesicles. A similar change in the distribution of the reduction phases was observed upon decreasing the phosphatidylcholine content of the vesicles; the fast phase reduction being more pronounced in membranes with higher relative amounts of the protein components. A decrease of the rate of O-demethylation of p-nitroanisole catalyzed by P-450 LM2 parallel to the extent of fast phase reduction was observed upon dilution of neutral phosphatidylcholine membranes with phospholipid. By contrast, no effect of lipid dilution was evident in negatively charged membranes. The results are consistent with the hypothesis that the extent of fast phase reduction is governed by the amount of complex formed between NADPH-cytochrome P-450 reductase and cytochrome P-450 in the membranes; negative membranes appear to favor the formation of such complexes, whereas similar complexes are less formed, or are not functional, in neutral membranes.
研究了在重构磷脂囊泡中由NADPH - 细胞色素P - 450还原酶介导的细胞色素P - 450 LM2还原的动力学。观察到在磷脂酰胆碱囊泡中血红素蛋白的还原效率较低。然而，通过将带负电荷的磷脂引入膜中，还原速率随着囊泡产生的净负电荷而相应增加。在存在苄非他明的情况下，向系统中添加NADPH后1秒时细胞色素P - 450 LM2的还原程度是所用囊泡电泳迁移率的线性函数。在没有底物的情况下，通过电泳测量的囊泡净负电荷与还原速率之间也获得了类似的关系。增强的还原主要反映在还原相分布的改变上；在不存在或存在添加底物的情况下快速相还原的程度取决于囊泡的电泳迁移率。当降低囊泡中磷脂酰胆碱的含量时，观察到还原相分布有类似变化；在蛋白质成分相对含量较高的膜中快速相还原更明显。在用磷脂稀释中性磷脂酰胆碱膜时，观察到由P - 450 LM2催化的对硝基苯甲醚O - 去甲基化速率的降低与快速相还原程度平行。相比之下，在带负电荷的膜中脂质稀释没有明显影响。这些结果与以下假设一致，即快速相还原的程度由膜中NADPH - 细胞色素P - 450还原酶和细胞色素P - 450之间形成的复合物的量决定；带负电荷的膜似乎有利于这种复合物的形成，而在中性膜中类似的复合物形成较少或没有功能。
