Functional Interactions between Cytochromes P450 1A2 and 2B4 Require Both Enzymes to Reside in the Same Phospholipid Vesicle EVIDENCE FOR PHYSICAL COMPLEX FORMATION
Functional Interactions between Cytochromes P450 1A2 and 2B4 Require Both Enzymes to Reside in the Same Phospholipid Vesicle EVIDENCE FOR PHYSICAL COMPLEX FORMATION
复制标题
DOI：
10.1074/jbc.m109.076885
复制
发表时间：
2010-03-19
影响因子：
4.8
通讯作者：
Backes, Wayne L.
中科院分区：
文献类型：
--
作者：
Reed, James R.;Eyer, Marilyn;Backes, Wayne L.
先前的研究已经表明，两种细胞色素P450酶（P450）的组合存在可以影响两种酶的功能，结果与异聚体P450.P450复合物的形成一致。本研究的目的是通过确定P450 1A 2（CYP 1A 2）和P450 2B 4（CYP 2B 4）之间的相互作用是否需要两种酶驻留在相同的脂质囊泡中，为P450 1A 2（CYP 1A 2）和P450 2B 4（CYP 2B 4）之间的物理相互作用提供直接证据。当NADPH-细胞色素P450还原酶（CPR）和一个单一的P450被纳入单独的囊泡，观察到非常缓慢的还原速率，表明酶锚定在囊泡。接下来，制备几种复溶系统：1）CPR.CYP1A2，2）CPR.CYP2B4，3）CPR.CYP1A2囊泡与CPR.CYP2B4囊泡的混合物，和4）相同囊泡中的CPR.CYP1A2.CYP2B4（三元系统）。当在三元系统中时，CYP 2B 4介导的代谢被显著抑制，并且CYP 1A 2活性被替代P450的存在刺激。相反，P450在单独的囊泡中不能相互作用。这些数据表明，P450必须在相同的囊泡改变代谢。通过与双（磺基琥珀酰亚胺基）辛二酸酯交联，提供了CPR、CYP 1A 2和CYP 2B 4之间物理相互作用的其他证据。结果表明，交联后，CYP 1A 2抗体能够免疫共沉淀CYP 2B 4，但只有当两种蛋白质在相同的磷脂囊泡。这些结果清楚地表明，P450功能的改变需要两个P450存在于相同的囊泡中，并支持P450在膜中形成物理复合物的机制。
Previous studies have shown that the combined presence of two cytochrome P450 enzymes (P450s) can affect the function of both enzymes, results that are consistent with the formation of heteromeric P450.P450 complexes. The goal of this study was to provide direct evidence for a physical interaction between P450 1A2 (CYP1A2) and P450 2B4 (CYP2B4), by determining if the interactions required both enzymes to reside in the same lipid vesicles. When NADPH-cytochrome P450 reductase (CPR) and a single P450 were incorporated into separate vesicles, extremely slow reduction rates were observed, demonstrating that the enzymes were anchored in the vesicles. Next, several reconstituted systems were prepared: 1) CPR.CYP1A2, 2) CPR.CYP2B4, 3) a mixture of CPR.CYP1A2 vesicles with CPR.CYP2B4 vesicles, and 4) CPR.CYP1A2.CYP2B4 in the same vesicles (ternary system). When in the ternary system, CYP2B4-mediated metabolism was significantly inhibited, and CYP1A2 activities were stimulated by the presence of the alternate P450. In contrast, P450s in separate vesicles were unable to interact. These data demonstrate that P450s must be in the same vesicles to alter metabolism. Additional evidence for a physical interaction among CPR, CYP1A2, and CYP2B4 was provided by cross-linking with bis(sulfosuccinimidyl) suberate. The results showed that after cross-linking, antibody to CYP1A2 was able to co-immunoprecipitate CYP2B4 but only when both proteins were in the same phospholipid vesicles. These results clearly demonstrate that the alterations in P450 function require both P450s to be present in the same vesicles and support a mechanism whereby P450s form a physical complex in the membrane.