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Research ArticleArticle

Relationship between CYP1A2 Localization and Lipid Microdomain Formation as a Function of Lipid Composition

Lauren M. Brignac-Huber, James R. Reed, Marilyn K. Eyer and Wayne L. Backes
Drug Metabolism and Disposition November 2013, 41 (11) 1896-1905; DOI: https://doi.org/10.1124/dmd.113.053611
Lauren M. Brignac-Huber
Department of Pharmacology and Experimental Therapeutics, Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, New Orleans, Louisiana
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James R. Reed
Department of Pharmacology and Experimental Therapeutics, Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, New Orleans, Louisiana
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Marilyn K. Eyer
Department of Pharmacology and Experimental Therapeutics, Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, New Orleans, Louisiana
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Wayne L. Backes
Department of Pharmacology and Experimental Therapeutics, Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, New Orleans, Louisiana
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Abstract

Cytochrome P450 (P450) function requires the interaction of P450 and NADPH-cytochrome P450 reductase (CPR) in membranes, and is frequently studied using reconstituted systems composed solely of phosphatidylcholine. There is increasing evidence that other endoplasmic reticulum (ER) lipids can affect P450 structure, activity, and interactions with CPR. Some of these lipid effects have been attributed to the formation of organized liquid-ordered (lo) domains. The goal of this study was to determine if lo domains were formed in P450 reconstituted systems mimicking the ER membrane. CYP1A2, when incorporated in “ER-like” lipid vesicles, displayed detergent insolubility after treatment with Brij 98 and centrifugation in a sucrose gradient. Lipid probes were employed to identify domain formation in both ER-like vesicles and model membranes known to form lo domains. Changes in fluorescence resonance energy transfer (FRET) using an established donor/acceptor FRET pair in both ER-like and model lo-forming systems demonstrated the coexistence of lo- and liquid-disordered domains as a function of cholesterol and sphingomyelin content. Similarly, 6-dodecanoyl-2-dimethylaminonaphthalene (laurdan), a probe that reports on membrane organization, showed that cholesterol and sphingomyelin increased membrane order. Finally, brominated-phosphatidylcholine allowed for monitoring of the location of both CPR and CYP1A2 within the lo regions of ER-like systems. Taken together, the results demonstrate that ER-like vesicles generate microdomains, and both CYP1A2 and CPR predominantly localize into lo membrane regions. Probe fluorescent responses suggest that lipid microdomains form in these vesicles whether or not enzymes are included in the reconstituted systems. Thus, it does not appear that the proteins are critical for stabilizing lo domains.

Footnotes

    • Received July 11, 2013.
    • Accepted August 20, 2013.
  • L.M.B.-H. and J.R.R. contributed equally to the preparation of this manuscript and are designated as co-first authors.

  • This work was supported by a US Public Health Services research grant from the National Institutes of Health National Institute of Environmental Health Sciences [Grant R01 ES004344]; and a predoctoral research fellowship from the State of Louisiana Board of Regents [Grant LEQSF(2005-10) GF-08].

  • dx.doi.org/10.1124/dmd.113.053611.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

  • Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 41 (11)
Drug Metabolism and Disposition
Vol. 41, Issue 11
1 Nov 2013
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Research ArticleArticle

CYP1A2 Localization in Microdomains of Lipid Vesicles

Lauren M. Brignac-Huber, James R. Reed, Marilyn K. Eyer and Wayne L. Backes
Drug Metabolism and Disposition November 1, 2013, 41 (11) 1896-1905; DOI: https://doi.org/10.1124/dmd.113.053611

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Research ArticleArticle

CYP1A2 Localization in Microdomains of Lipid Vesicles

Lauren M. Brignac-Huber, James R. Reed, Marilyn K. Eyer and Wayne L. Backes
Drug Metabolism and Disposition November 1, 2013, 41 (11) 1896-1905; DOI: https://doi.org/10.1124/dmd.113.053611
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