title: Relationship between CYP1A2 localization and lipid microdomain formation as a function of lipid composition.
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 (l(o)) domains. The goal of this study was to determine if l(o) 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 l(o) domains. Changes in fluorescence resonance energy transfer (FRET) using an established donor/acceptor FRET pair in both ER-like and model l(o)-forming systems demonstrated the coexistence of l(o)- 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 l(o) regions of ER-like systems. Taken together, the results demonstrate that ER-like vesicles generate microdomains, and both CYP1A2 and CPR predominantly localize into l(o) 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 l(o) domains.
authors: Department of Pharmacology and Experimental Therapeutics, Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, New Orleans, Louisiana.
authors: Lauren M
authors: LM
authors: Brignac-Huber
authors: James R
authors: JR
authors: Reed
authors: Marilyn K
authors: MK
authors: Eyer
authors: Wayne L
authors: WL
authors: Backes
mesh_terms: D000818:Animals; D002417:Cattle; D002645:Chickens; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D008055:Lipids; D021962:Membrane Microdomains / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D013552:Swine
chemical_list: D008055:Lipids; D010743:Phospholipids; D003577:Cytochrome P-450 Enzyme System; D019388:Cytochrome P-450 CYP1A2
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