1: Biochemistry  1983 Jul 19;22(15):3586-94 

Possible association of NADPH-cytochrome P-450 reductase and cytochrome P-450 in
reconstituted phospholipid vesicles.

Nisimoto Y, Kinosita K Jr, Ikegami A, Kawai N, Ichihara I, Shibata Y.

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.

PMID: 6412745 [PubMed - indexed for MEDLINE]