Dutton D R, McMillen S K, Sonderfan A J, Thomas P E, Parkinson A
Arch Biochem Biophys. 1987 Jun;255(2):316-28. doi: 10.1016/0003-9861(87)90399-7.
The aim of the present study was to examine a recent proposal that inhibitory isozyme:isozyme interactions explain why membrane-bound isozymes of rat liver microsomal cytochrome P-450 exert only a fraction of the catalytic activity they express when purified and reconstituted with saturating amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. The different pathways of testosterone hydroxylation catalyzed by cytochromes P-450a (7 alpha-hydroxylation), P-450b (16 beta-hydroxylation), and P-450c (6 beta-hydroxylation) enabled possible inhibitory interactions between these isozymes to be investigated simultaneously with a single substrate. No loss of catalytic activity was observed when purified cytochromes P-450a, P-450b, or P-450c were reconstituted in binary or ternary mixtures under a variety of incubation conditions. When purified cytochromes P-450a, P-450b, and P-450c were reconstituted under conditions that mimicked a microsomal system (with respect to the absolute concentration of both the individual cytochrome P-450 isozyme and NADPH-cytochrome P-450 reductase), their catalytic activity was actually less (69-81%) than that of the microsomal isozymes. These results established that cytochromes P-450a, P-450b, and P-450c were not inhibited by each other, nor by any of the other isozymes in the liver microsomal preparation. Incorporation of purified NADPH-cytochrome P-450 reductase into liver microsomes from Aroclor 1254-induced rats stimulated the catalytic activity of cytochromes P-450a, P-450b, and P-450c. Similarly, purified cytochromes P-450a, P-450b, and P-450c expressed increased catalytic activity in a reconstituted system only when the ratio of NADPH-cytochrome P-450 reductase to cytochrome P-450 exceeded that normally found in liver microsomes. These results indicate that the inhibitory cytochrome P-450 isozyme:isozyme interactions described for warfarin hydroxylation were not observed when testosterone was the substrate. In addition to establishing that inhibitory interactions between different cytochrome P-450 isozymes is not a general phenomenon, the results of the present study support a simple mass action model for the interaction between membrane-bound or purified cytochrome P-450 and NADPH-cytochrome P-450 reductase during the hydroxylation of testosterone.
本研究的目的是检验最近的一项提议，即抑制性同工酶：同工酶相互作用解释了为什么大鼠肝微粒体细胞色素P - 450的膜结合同工酶在与饱和量的NADPH - 细胞色素P - 450还原酶和最佳量的二月桂酰磷脂酰胆碱纯化并重构时，仅发挥其表达的催化活性的一部分。细胞色素P - 450a（7α - 羟基化）、P - 450b（16β - 羟基化）和P - 450c（6β - 羟基化）催化的睾酮羟基化的不同途径，使得这些同工酶之间可能的抑制性相互作用能够用单一底物同时进行研究。当在各种孵育条件下将纯化的细胞色素P - 450a、P - 450b或P - 450c在二元或三元混合物中重构时，未观察到催化活性的损失。当在模拟微粒体系统的条件下（就单个细胞色素P - 450同工酶和NADPH - 细胞色素P - 450还原酶的绝对浓度而言）将纯化的细胞色素P - 450a、P - 450b和P - 450c重构时，它们的催化活性实际上比微粒体同工酶的活性低（69 - 81%）。这些结果表明，细胞色素P - 450a、P - 450b和P - 450c彼此之间不受抑制，也不受肝微粒体制剂中任何其他同工酶的抑制。将纯化的NADPH - 细胞色素P - 450还原酶掺入来自Aroclor 1254诱导大鼠的肝微粒体中，刺激了细胞色素P - 450a、P - 450b和P - 450c的催化活性。同样，仅当NADPH - 细胞色素P - 450还原酶与细胞色素P - 450的比例超过肝微粒体中通常发现的比例时，纯化的细胞色素P - 450a、P - 450b和P - 450c在重构系统中才表现出增加的催化活性。这些结果表明，当睾酮作为底物时，未观察到华法林羟基化中描述的抑制性细胞色素P - 450同工酶：同工酶相互作用。除了确定不同细胞色素P - 450同工酶之间的抑制性相互作用不是普遍现象外，本研究的结果支持了在睾酮羟基化过程中膜结合或纯化的细胞色素P - 450与NADPH - 细胞色素P - 450还原酶之间相互作用的简单质量作用模型。
