Wagner S L, Dean W L, Gray R D
Biochemistry. 1987 Apr 21;26(8):2343-8. doi: 10.1021/bi00382a040.
Hydroxylation of acetanilide catalyzed by purified cytochrome P-450LM4 and NADPH-cytochrome P-450 reductase was reconstituted with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The optimum rate of production of 4-hydroxyacetanilide was observed between 3 and 7 mM CHAPS and was about half that with 0.05 mM dilauroylglyceryl-3-phosphocholine (di-12-GPC). At higher detergent concentrations, hydroxylase activity decreased until at 15-20 mM CHAPS the system was inactive. The effect of CHAPS on the state of aggregation of P-450LM4 and on interaction between the cytochrome and P-450 reductase alone and under turnover conditions was investigated by ultracentrifugation. At 4 mM CHAPS, P-450LM4 was hexameric to heptameric (Mr 369,000). Neither reductase nor reductase plus acetanilide and NADPH altered the state of P-450LM4 aggregation, suggesting that a stable 1:1 P-450/reductase complex did not form under turnover conditions. Replacing CHAPS with 0.05 mM di-12-GPC resulted in formation of heterogeneous P-450 oligomers (Mr greater than 480,000). At CHAPS concentrations where substrate hydroxylation did not occur (15 and 22 mM), P-450LM4 was shown by sedimentation equilibrium measurements to be dimeric and monomeric, respectively. P-450 reductase was shown to reduce monomeric P-450LM4 in the presence of NADPH. Thus, the dependence of hydroxylase activity on [CHAPS] may be related to the state of aggregation of the cytochrome. An apparent correlation between P-450 aggregation state and NADPH-supported hydroxylation was also observed with phenobarbital-inducible P-450LM2 in the presence of detergents [Dean, W.L., & Gray, R.D. (1982) J. Biol. Chem. 257, 14679-14685; Wagner, S.L., Dean, W.L., & Gray, R.D. (1984) J. Biol. Chem. 259, 2390-2395].
纯化的细胞色素P-450LM4和NADPH-细胞色素P-450还原酶催化乙酰苯胺的羟基化反应，用两性离子去污剂3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸盐（CHAPS）进行了重组。在3至7 mM CHAPS之间观察到4-羟基乙酰苯胺的最佳生成速率，约为0.05 mM二月桂酰甘油-3-磷酸胆碱（二-12-GPC）时的一半。在更高的去污剂浓度下，羟化酶活性降低，直到在15至20 mM CHAPS时系统失活。通过超速离心研究了CHAPS对P-450LM4聚集状态以及细胞色素与P-450还原酶单独存在和在周转条件下相互作用的影响。在4 mM CHAPS时，P-450LM4为六聚体至七聚体（Mr 369,000）。还原酶以及还原酶加乙酰苯胺和NADPH均未改变P-450LM4的聚集状态，这表明在周转条件下未形成稳定的1:1 P-450/还原酶复合物。用0.05 mM二-12-GPC替代CHAPS导致形成异质P-450寡聚体（Mr大于480,000）。在底物羟基化未发生的CHAPS浓度（15和22 mM）下，沉降平衡测量显示P-450LM4分别为二聚体和单体。在NADPH存在下，P-450还原酶显示可还原单体P-450LM4。因此，羟化酶活性对[CHAPS]的依赖性可能与细胞色素的聚集状态有关。在去污剂存在的情况下，对于苯巴比妥诱导的P-450LM2，也观察到了P-450聚集状态与NADPH支持的羟基化之间的明显相关性[迪恩，W.L.，&格雷，R.D.（1982年）《生物化学杂志》257，14679 - 14685；瓦格纳，S.L.，迪恩，W.L.，&格雷，R.D.（1984年）《生物化学杂志》259，2390 - 2395]。
