Effect of microsome-liposome fusion on the rotational mobility of cytochrome P450IIB4 in rabbit liver microsomes

Membrane fusion of microsomes with soybean phospholipid vesicles was perfor med at pH 6.5 to investigate the effect of lipid-enrichment in the membrane on the rotational mobility of cytochrome P450. Rotational diffusion of cyt ochrome P450 in the microsomal membrane of phenobarbital-induced rabbit liv er was measured by detecting the decay of absorption anisotropy after photo lysis of the heme CO complex by a vertically polarized laser Rash, The fusi on procedures yielded three separate fractions upon sucrose density gradien t 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 fr action. In each fraction. co-existence of mobile and immobile cytochrome: P 450 was observed. The percentage of rotationally mobile P450 (with the mean rotational relaxation time of phi =505-828 muS) in each of the different b ands 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 d ue to lipid-enrichment indicates that aggregated proteins in microsomal mem branes dissociate with increasing L/P which is inversely proportional to th e protein concentration in the membrane. With freeze-fracture electron micr oscopy, it was shown that the average distance increased between intramembr ane panicles by lipid-enrichment. Thus. the significant immobile population (32%) of P450 in microsomal membranes can be explained by nonspecific prot ein 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 micros omes was shown to be due to the pH 6.5 incubation used for fusion. (C) 2001 Elsevier Science B.V. All rights reserved.