Kawato S, Lehner C, Müller M, Cherry R J
J Biol Chem. 1982 Jun 10;257(11):6470-6.
Rotational diffusion of cytochrome oxidase in the inner membrane of rat liver mitochondria was measured by detecting the decay of absorption anisotropy after photolysis of the heme a3.CO complex by a vertically polarized laser flash. As in previous experiments with beef heart mitochondria (Kawato, S., Sigel, E., Carafoli, E., and Cherry, R. J. (1980) J. Biol. Chem. 255, 5508-5510), co-existence of rotating cytochrome oxidase (mean rotational relaxation time, phi, of 700 to 1400 microseconds) and immobilized cytochrome oxidase (phi greater than 20 ms) was observed in mitochondria and mitoplasts. The effect of lipid/protein ratio by weight (L/P) on the relative proportions of mobile and immobile cytochrome oxidase was investigated following the fusion of soybean phospholipid vesicles with mitoplasts. The fusion procedure yielded four separate fractions upon sucrose density gradient centrifugation with L/P as follows: 0.3 in Pellet, 0.7 in Band 3, 1.5 in Band 2, and 3.0 in Band 1. The percentage of rotationally mobile cytochrome oxidase (phi = 700 to 1000 microseconds) in each of the different bands was found to be 16% in Pellet, 25% in Band 3, 47% in Band 2, and 76% in Band 1 at 37 degrees C. The dependence of the amount of mobile cytochrome oxidase on L/P indicates that the fraction of aggregated protein progressively decreases with decreasing concentration of proteins in the membrane. Thus, the large immobile fraction of cytochrome oxidase in mitochondrial inner membranes can be explained by nonspecific protein aggregation which is a consequence of the low L/P. The decrease in the mobile fraction in Pellet compared with mitoplasts was shown to be due to the pH 6.5 incubation used for fusion.
通过检测垂直偏振激光闪光使血红素a3 - CO复合物光解后吸收各向异性的衰减，来测定大鼠肝脏线粒体内膜中细胞色素氧化酶的旋转扩散。如同先前对牛心线粒体所做的实验（河户，S.，西格尔，E.，卡拉福里，E.，以及彻里，R. J.（1980年）《生物化学杂志》255卷，5508 - 5510页），在线粒体和线粒体小体中观察到了旋转的细胞色素氧化酶（平均旋转弛豫时间φ为700至1400微秒）和固定化的细胞色素氧化酶（φ大于20毫秒）共存。在大豆磷脂囊泡与线粒体小体融合后，研究了重量脂质/蛋白质比（L/P）对可移动和不可移动细胞色素氧化酶相对比例的影响。融合过程在蔗糖密度梯度离心后产生了四个单独的组分，L/P如下：沉淀中为0.3，条带3中为0.7，条带2中为1.5，条带1中为3.0。发现在37℃时，不同条带中旋转可移动细胞色素氧化酶（φ = 700至1000微秒）的百分比在沉淀中为16%，条带3中为25%，条带2中为47%，条带1中为76%。可移动细胞色素氧化酶的量对L/P的依赖性表明，随着膜中蛋白质浓度降低，聚集蛋白的比例逐渐减少。因此，线粒体内膜中细胞色素氧化酶的大量不可移动部分可以用非特异性蛋白质聚集来解释，这是低L/P的结果。与线粒体小体相比，沉淀中可移动部分的减少被证明是由于融合所用的pH 6.5孵育。
