Yamada M, Ohta Y, Bachmanova G I, Archakov A I, Hatta I, Kawato S
Department of Biophysics and Life Sciences, Graduate School of Arts and Sciences, University of Tokyo at Komaba, Meguro, Tokyo, Japan.
J Inorg Biochem. 2001 Feb;83(4):261-8. doi: 10.1016/s0162-0134(00)00169-0.
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.
在pH 6.5条件下进行微粒体与大豆磷脂囊泡的膜融合，以研究膜中脂质富集对细胞色素P450旋转流动性的影响。通过检测血红素CO复合物经垂直偏振激光闪光光解后吸收各向异性的衰减，来测量苯巴比妥诱导的兔肝微粒体膜中细胞色素P450的旋转扩散。融合程序经蔗糖密度梯度离心后产生三个单独的组分，其重量脂质与蛋白质比（L/P）如下：底部组分中为1.5，中间组分中为2.2，顶部组分中为3.9。在每个组分中，均观察到了可移动和不可移动的细胞色素P450共存。发现在不同条带中，旋转可移动的P450（平均旋转弛豫时间为φ=505 - 828微秒）的百分比在底部组分中为59%，在中间组分中为61%，在顶部组分中为68%。由于脂质富集导致的P450可移动群体的增加表明，微粒体膜中的聚集蛋白随着L/P的增加而解离，L/P与膜中蛋白质浓度成反比。通过冷冻蚀刻电子显微镜观察发现，脂质富集使膜内颗粒之间的平均距离增加。因此，微粒体膜中P450显著的不可移动群体（32%）可由非特异性蛋白质聚集来解释，这是低L/P（0.8）的结果。与完整微粒体相比，底部组分中可移动群体的减少被证明是由于融合所用的pH 6.5孵育所致。
