Abstract
Cytochromes P450 (P450s) interact with redox transfer proteins, including P450 reductase (CPR) and cytochrome b5 (b5), all being membrane-bound. In multiple in vitro systems, P450-P450 interactions also have been observed, resulting in alterations in enzymatic activity. The current work investigated the effects and mechanisms of interaction between CYP2C9 and CYP3A4 in a reconstituted system. CYP2C9-mediated metabolism of S-naproxen and S-flurbiprofen was inhibited up to 80% by coincubation with CYP3A4, although Km values were unchanged. Increasing CYP3A4 concentrations increased the degree of inhibition, whereas increasing CPR concentrations resulted in less inhibition. Addition of b5 only marginally affected the magnitude of inhibition. In contrast, CYP2C9 did not alter the CYP3A4-mediated metabolism of testosterone. The potential role of the hydrophobic N terminus on these interactions was assessed by incubating truncated CYP2C9 with full-length CYP3A4, and vice versa. In both cases, the inhibition was fully abolished, indicating an important role for hydrophobic forces in CYP2C9-CYP3A4 interactions. Finally, a CYP2C9/CYP3A4 heteromer complex was isolated by coimmunoprecipitation techniques, confirming the physical interaction of the proteins. These results show that the N-terminal membrane binding domains of CYP2C9 and CYP3A4 are involved in heteromer complex formation and that at least one consequence is a reduction in CYP2C9 activity.
Footnotes
This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant GM063215, GM086891].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.109.030155.
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ABBREVIATIONS:
- P450
- cytochrome P450
- CPR
- cytochrome P450 reductase
- b5
- cytochrome b5
- DOPC
- l-α-dioleoyl-sn-gly-cero-3-phosphocholine
- TBST
- Tris-buffered saline/Tween
- DHT
- dihydrotestosterone
- TST
- testosterone
- rCPR
- rat cytochrome P450 reductase
- hCPR
- human cytochrome P450 reductase
- CYP2C9(t)
- truncated CYP2C9
- CYP3A4(t)
- truncated CYP3A4.
- Received September 4, 2009.
- Accepted March 9, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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