Department of Pharmacology, Stanley S Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
J Biol Chem. 2010 Mar 19;285(12):8942-52. doi: 10.1074/jbc.M109.076885. Epub 2010 Jan 13.
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.
先前的研究表明，两种细胞色素 P450 酶（P450s）的共同存在会影响两种酶的功能，这与形成异源 P450.P450 复合物的结果一致。本研究的目的是通过确定相互作用是否需要两种酶存在于同一脂质体中来提供 P450 1A2（CYP1A2）和 P450 2B4（CYP2B4）之间物理相互作用的直接证据。当将 NADPH-细胞色素 P450 还原酶（CPR）和单一 P450 掺入到单独的囊泡中时，观察到极其缓慢的还原速率，这表明酶被锚定在囊泡中。接下来，制备了几种重建系统：1）CPR.CYP1A2，2）CPR.CYP2B4，3）CPR.CYP1A2 囊泡与 CPR.CYP2B4 囊泡的混合物，以及 4）同一囊泡中的 CPR.CYP1A2.CYP2B4（三元系统）。在三元系统中，CYP2B4 介导的代谢明显受到抑制，而替代 P450 的存在会刺激 CYP1A2 的活性。相比之下，单独囊泡中的 P450 无法相互作用。这些数据表明 P450 必须在同一囊泡中才能改变代谢。CPR、CYP1A2 和 CYP2B4 之间的物理相互作用的其他证据是通过使用双（磺基琥珀酰亚胺基）丁二酸酯交联提供的。结果表明，交联后，CYP1A2 的抗体能够共免疫沉淀 CYP2B4，但仅当两种蛋白质都存在于同一磷脂囊泡中时才如此。这些结果清楚地表明，P450 功能的改变需要两种 P450 都存在于同一囊泡中，并支持一种机制，即 P450 在膜中形成物理复合物。
