title: Effect of microsome-liposome fusion on the rotational mobility of cytochrome P450IIB4 in rabbit liver microsomes.
abstract: Membrane fusion of microsomes with soybean phospholipid vesicles was performed at pH 6.5 to investigate the effect of lipid-enrichment in the membrane on the rotational mobility of cytochrome P450. Rotational diffusion of cytochrome P450 in the microsomal membrane of phenobarbital-induced rabbit liver was measured by detecting the decay of absorption anisotropy after photolysis of the heme CO complex by a vertically polarized laser flash. The fusion procedures yielded three separate fractions upon sucrose density gradient centrifugation with lipid-to-protein ratio in weight (L/P) as follows: 1.5 in the bottom fraction, 2.2 in the middle fraction, and 3.9 in the top fraction. In each fraction, co-existence of mobile and immobile cytochrome P450 was observed. The percentage of rotationally mobile P450 (with the mean rotational relaxation time of phi=505-828 micros) in each of the different bands was found to be 59% in the bottom fraction, 61% in the middle fraction, and 68% in the top fraction. This increase in mobile population of P450 due to lipid-enrichment indicates that aggregated proteins in microsomal membranes dissociate with increasing L/P which is inversely proportional to the protein concentration in the membrane. With freeze-fracture electron microscopy, it was shown that the average distance increased between intramembrane particles by lipid-enrichment. Thus, the significant immobile population (32%) of P450 in microsomal membranes can be explained by nonspecific protein aggregation which is a consequence of the low L/P of 0.8. The decrease in the mobile population in the bottom fraction compared with intact microsomes was shown to be due to the pH 6.5 incubation used for fusion.
authors: Department of Biophysics and Life Sciences, Graduate School of Arts and Sciences, University of Tokyo at Komaba, Meguro, Tokyo, Japan.
authors: M
authors: M
authors: Yamada
authors: Y
authors: Y
authors: Ohta
authors: G I
authors: GI
authors: Bachmanova
authors: A I
authors: AI
authors: Archakov
authors: I
authors: I
authors: Hatta
authors: S
authors: S
authors: Kawato
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D005614:Freeze Fracturing; D007425:Intracellular Membranes / Q000201:enzymology / Q000648:ultrastructure; D007700:Kinetics; D008081:Liposomes; D008297:Male; D008561:Membrane Fusion; D008862:Microsomes, Liver / Q000201:enzymology* / Q000648:ultrastructure; D011817:Rabbits; D012399:Rotation; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D008081:Liposomes; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C050356:steroid 15-alpha-hydroxylase