The effect of NADPH concentration on the reduction of cytochrome P-450 LM2.
The effect of NADPH concentration on the reduction of cytochrome P-450 LM2.
复制标题
NADPH浓度对细胞色素P-450 LM2还原的影响。
DOI：
10.1016/s0021-9258(19)57385-9
复制
发表时间：
1988
期刊：
影响因子：
--
通讯作者：
C. E. Reker
中科院分区：
文献类型：
--
作者：
W. Backes;C. E. Reker
在没有底物和1mm苯非他明存在的情况下，在一系列NADPH浓度下测量细胞色素P-450 LM2的还原。在没有底物还原的情况下，可以描述为一个双相过程，55%的反应发生在第一阶段（在20微米NADPH下）。当苯丙胺存在时，在第一阶段发生的反应比例增加到91%。在不存在或不存在苯丙胺的情况下进行检测时，随着NADPH浓度的降低，快速相中降低的速率常数和LM2的比例也随之降低。在每一种情况下，在第二阶段减少的LM2的比例在检查NADPH浓度时没有实质性的改变。为了解释NADPH浓度对LM2还原初始速率的影响，我们考察了NADPH对NADPH-细胞色素P-450还原酶还原的影响。由于在每个还原酶分子中存在两种黄素，因此在给定的NADPH浓度下，还原酶可能存在九种可能的氧化还原状态。根据黄素半反应和NADPH氧化的氧化还原电位，可以确定每个还原酶亚种的相对浓度。研究了不同还原酶亚种对LM2还原的速率常数，并将不同NADPH浓度下LM2还原的理论初始速率与实验值进行了比较。实验数据与模型一致，在本试验条件下，完全还原的还原酶是主要负责LM2还原的形式。
Cytochrome P-450 LM2 reduction was measured at a series of NADPH concentrations in the absence of substrate and in the presence of 1 mM benzphetamine. In the absence of substrate reduction could be described as a biphasic process with 55% of the reaction occurring in the first phase (at 20 microM NADPH). When benzphetamine was present, the fraction of the reaction occurring in the first phase was increased to 91%. When examined either in the absence or presence of benzphetamine, the rate constant and fraction of LM2 reduced in the fast phase were decreased as the NADPH concentration was decreased. In each case the fraction of LM2 reduced in the second phase was not substantially altered over the NADPH concentrations examined. To explain the effect of NADPH concentration on the initial rate of LM2 reduction, the effect of NADPH on the reduction of NADPH-cytochrome P-450 reductase was examined. Due to the presence of two flavins within each reductase molecule, there would be nine possible oxidation-reduction states of the reductase which may be present at a given NADPH concentration. Based on the redox potentials for the flavin half-reactions and for NADPH oxidation, the relative concentrations of each of the reductase subspecies could be determined. Rate constants were assigned for the reduction of LM2 by the various reductase subspecies, and the theoretical initial rates of LM2 reduction at various NADPH concentrations were compared with values obtained experimentally. The experimental data are consistent with a model where, under the conditions of this assay, the fully reduced reductase is the form primarily responsible for the reduction of LM2.