Decreased lipid order induced by microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in model membranes: fluorescence and electron spin resonance studies.
Cytochrome P-450 and NADPH-cytochrome P-450 reductase were reconstituted in unilamellar lipid vesicles prepared by the cholate dialysis technique from pure dimyristoylphosphatidylcholine (DMPC), pure dipalmitoylphosphatidylcholine (DPPC), pure dioleoylphosphatidylcholine (DOPC), and phosphatidylcholine/phosphatidylethanolamine/phosphatidylserine (PC/PE/PS) (10:5:1). As probes for the vesicles' hydrocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) and spin-labeled PC were used. The steady-state and time-resolved fluorescence parameters of DPH were determined as a function of temperature and composition of liposomes. Incorporation of either protein alone or together increased the steady-state fluorescence anisotropy (rs) of DPH in DOPC and PC/PE/PS (10:5:1) liposomes. In DMPC and DPPC vesicles, the proteins decreased rs significantly below the transition temperature (Tc) of the gel to liquid-crystalline phase transition. Time-resolved fluorescence measurements of DPH performed in reconstituted PC/PE/PS and DMPC proteoliposomes showed that the proteins disorder the bilayer both in the gel and in the liquid-crystalline phase. Little disordering by the proteins was observed by a spin-label located near the mid-zone of the bilayer 1-palmitoyl-2-(5-doxylstearoyl)-3-sn-phosphatidylcholine (8-doxyl-PC), whereas pronounced disordering was detected by 1-palmitoyl-2-(8-doxylpalmitoyl)-3-sn-phosphatidylcholine (5-doxyl-PC), which probes the lipid zone closer to the polar part of the membrane. Fluorescence lifetime measurements of DPH indicate an average distance of greater than or equal to 60 A between the heme of cytochrome P-450 and DPH.
Kunz
B C
BC
Rehorek
M
M
Hauser
H
H
Winterhalter
K H
KH
Richter
C
C
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004134:Dimyristoylphosphatidylcholine; D004578:Electron Spin Resonance Spectroscopy; D005454:Fluorescence Polarization; D007700:Kinetics; D008081:Liposomes; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010713:Phosphatidylcholines; D011510:Proteolipids / Q000378:metabolism; D011663:Pulmonary Surfactants; D051381:Rats; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D008081:Liposomes; D010713:Phosphatidylcholines; D011510:Proteolipids; D011663:Pulmonary Surfactants; C020485:proteoliposomes; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C017251:1,2-oleoylphosphatidylcholine; D004134:Dimyristoylphosphatidylcholine