Kelley Rusty W, Cheng Dongmei, Backes Wayne L
Department of Pharmacology and Experimental Therapeutics and The Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 533 Bolivar Street, New Orleans, Louisiana 70112, USA.
Biochemistry. 2006 Dec 26;45(51):15807-16. doi: 10.1021/bi061803n.
Mixed reconstituted systems containing CYP2B4, CYP1A2, and NADPH-cytochrome P450 reductase were previously shown to exhibit a dramatic inhibition of 7-pentoxyresorufin O-dealkylation (PROD) when compared to simple reconstituted systems containing reductase and a single P450 enzyme, results consistent with the formation of CYP1A2-CYP2B4 complexes where the reductase binds with high affinity to the CYP1A2 moiety of the complex. In this report, we provide evidence for an interaction between CYP1A2 and CYP2E1. Synergism of 7-ethoxyresorufin O-deethylation (EROD) and PROD was observed when these P450s were combined in mixed reconstituted systems at subsaturating reductase concentrations. Higher ionic strength attenuated the synergistic stimulation of both PROD and EROD in mixed reconstituted systems, consistent with disruption of heteromeric CYP2E1-CYP1A2 complexes. The effect of ionic strength was further examined as a function of reductase concentration. At lower ionic strength, there was a significant synergistic stimulation of EROD. This synergistic stimulation diminished with increasing reductase concentration, resulting in an additive response as reductase became saturating. Interestingly, at high ionic strength, the synergism of EROD in the mixed reconstituted system was not observed. In contrast, mixed reconstituted systems containing CYP2E1 and CYP2B4 did not provide evidence for the formation of these heteromeric P450-P450 complexes. The synergistic stimulation observed with the reductase-CYP1A2-CYP2E1 mixed reconstituted system is consistent with the formation of a CYP1A2-CYP2E1 complex. Taken together with the lack of a kinetically detectable interaction between CYP2B4 and CYP2E1, and the previously reported CYP1A2-CYP2B4 interaction, these results suggest that CYP1A2 may facilitate the formation of complexes with other P450 enzymes.
与含有还原酶和单一细胞色素P450酶的简单重组系统相比，先前已证明含有CYP2B4、CYP1A2和NADPH - 细胞色素P450还原酶的混合重组系统对7 - 戊氧基试卤灵O - 脱烷基化（PROD）具有显著抑制作用，该结果与CYP1A2 - CYP2B4复合物的形成一致，其中还原酶与复合物的CYP1A2部分具有高亲和力结合。在本报告中，我们提供了CYP1A2与CYP2E1之间相互作用的证据。当这些细胞色素P450在亚饱和还原酶浓度下组合于混合重组系统中时，观察到7 - 乙氧基试卤灵O - 脱乙基化（EROD）和PROD的协同作用。更高的离子强度减弱了混合重组系统中PROD和EROD的协同刺激作用，这与异源CYP2E1 - CYP1A2复合物的破坏一致。进一步研究了离子强度作为还原酶浓度函数的影响。在较低离子强度下，EROD有显著的协同刺激作用。随着还原酶浓度增加，这种协同刺激作用减弱，当还原酶达到饱和时导致加和反应。有趣的是，在高离子强度下，未观察到混合重组系统中EROD的协同作用。相反，含有CYP2E1和CYP2B4的混合重组系统未提供这些异源细胞色素P450 - 细胞色素P450复合物形成的证据。还原酶 - CYP1A2 - CYP2E1混合重组系统中观察到的协同刺激作用与CYP1A2 - CYP2E1复合物的形成一致。结合CYP2B4与CYP2E1之间缺乏动力学可检测的相互作用以及先前报道的CYP1A2 - CYP2B4相互作用，这些结果表明CYP1A2可能促进与其他细胞色素P450酶形成复合物。
