Purification of human cytochromes P450 and their interactions in vesicular lipid reconstituted systems

Abstract

P450s are membrane‐bound proteins found in the endoplasmic reticulum, requiring interaction with NADPH‐cytochrome P450 reductase (CPR). Previously, using rabbit enzymes, we demonstrated that one P450 can influence the behavior of another P450 by forming P450•P450 complexes. The goal of this study was to determine (1) if similar P450•P450 complexes are found with human P450s, and (2) how these interactions are affected by membrane lipids. Human CYP1A2 and CYP3A4 were incorporated into different reconstituted systems at subsaturating CPR, and the activity of CYP1A2 and CYP3A4 was examined. Substrate metabolism by both CYP1A2 and CYP3A4 were sensitive to alterations in lipid composition, and may affect interactions among P450s. These interactions were examined by comparing simple systems, containing CPR and a single P450, and mixed systems contain multiple P450s in the same vesicles. Interestingly, when CYP1A2 and CYP3A4 were present in the same vesicles, an inhibition of CYP3A4 function was observed, consistent with studies with rabbit P450s. The result of altered function of CYP3A4 with CYP1A2 is consistent with P450•P450 complex formation. (Supported by NIH R01 ES004344 & P42 ES013648)