Possible association of NADPH-cytochrome P-450 reductase and cytochrome P-450 in reconstituted phospholipid vesicles.
A fluorescent probe, N-(1-anilinonaphth-4-yl)-maleimide (ANM), was specifically labeled to SH group(s) in the hydrophilic moiety of NADPH-cytochrome P-450 reductase at a ratio of 1 +/- 0.1 ANM/mol of protein. The ANM-labeled reductase and P-450 were reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles in which all of the enzymes were functionally active. The reconstitution of the mixed-function oxidase system was found to be strongly dependent on both the lipid to protein molar ratio and phospholipid composition. The interactions of ANM-labeled reductase with P-450 in proteoliposomes were investigated by perturbation of the fluorescence of ANM. Upon incorporation of P-450 into the phospholipids vesicles (ANM-reductase/P-450/lipids identical to 1:1.4:800), a significant decrease of total fluorescence intensity and slight increase of emission anisotropy of ANM were observed. In the average fluorescence lifetime of ANM bound with reductase, an appreciable change was shown between the absence and presence of P-450 in the vesicles. These data provide clear evidence that significant molecular interactions occur between the two proteins in a membranous reconstituted system.
D000818:Animals; D007202:Indicators and Reagents; D007700:Kinetics; D008081:Liposomes; D008301:Maleimides; D008854:Microscopy, Electron; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010743:Phospholipids / Q000494:pharmacology*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D007202:Indicators and Reagents; D008081:Liposomes; D008301:Maleimides; D010743:Phospholipids; C010854:N-(1-anilinonaphthalyl)-4-maleimide; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase