Ohta Y, Mitani F, Ishimura Y, Yanagibashi K, Kawamura M, Kawato S
Institute of Physics, College of Arts and Sciences, University of Tokyo at Komaba.
J Biochem. 1990 Jan;107(1):97-104. doi: 10.1093/oxfordjournals.jbchem.a123020.
Rotation of cytochrome P-450 was examined in bovine adrenocortical mitochondria before and after an enzymatic transformation of cholesterol into pregnenolone by cytochrome P-450scc in the presence of malate. Rotational diffusion was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate substrate-dependent intermolecular interactions of cytochrome P-450 with other redox components. Rotational mobility of cytochrome P-450 was significantly dependent on the decrease in cholesterol content by side chain cleavage reaction catalyzed by cytochrome P-450scc. In a typical experiment, the observed value for the normalized time-independent anisotropy r(infinity)/r(0) was decreased from 0.78 in control mitochondria to 0.60 after conversion of 21% of cholesterol to pregnenolone, while no significant change was observed for the average rotational relaxation time phi of about 700 microseconds. Significantly high values of r(infinity)/r(0) = 0.78 and 0.60 imply co-existence of mobile and immobile populations of cytochrome P-450. Since we observed that the heme angle tilted 55 degrees from membrane plane, 22% (control mitochondria) and 40% (after conversion of cholesterol to pregnenolone) of cytochrome P-450 in mitochondria are calculated to be mobile in the preparation. The significant mobilization of cytochrome P-450scc molecules caused by the conversion of cholesterol to pregnenolone is likely due to changes in protein-protein interactions with its redox partners, since the lipid fluidity was kept unchanged by the cholesterol depletion.(ABSTRACT TRUNCATED AT 250 WORDS)
在苹果酸存在的情况下，通过细胞色素P-450scc将胆固醇酶促转化为孕烯醇酮之前和之后，对牛肾上腺皮质线粒体中细胞色素P-450的旋转进行了检测。通过观察血红素-CO复合物在垂直偏振激光闪光光解后吸收各向异性r(t)的衰减来测量旋转扩散。r(t)的分析基于“围绕膜法线旋转”模型。这些测量用于研究细胞色素P-450与其他氧化还原成分的底物依赖性分子间相互作用。细胞色素P-450的旋转流动性显著依赖于细胞色素P-450scc催化的侧链裂解反应导致的胆固醇含量降低。在一个典型实验中，归一化的与时间无关的各向异性r(∞)/r(0)的观测值从对照线粒体中的0.78降至21%的胆固醇转化为孕烯醇酮后的0.60，而约700微秒的平均旋转弛豫时间φ没有显著变化。r(∞)/r(0) = 0.78和0.60的显著高值意味着细胞色素P-450存在可移动和不可移动群体。由于我们观察到血红素角度相对于膜平面倾斜55度，计算得出线粒体中22%（对照线粒体）和40%（胆固醇转化为孕烯醇酮后）的细胞色素P-450在制剂中是可移动的。胆固醇转化为孕烯醇酮导致细胞色素P-450scc分子的显著移动可能是由于与其氧化还原伙伴的蛋白质-蛋白质相互作用发生了变化，因为胆固醇消耗后脂质流动性保持不变。（摘要截短于250字）
