Yamada M, Ohta Y, Bachmanova G I, Nishimoto Y, Archakov A I, Kawato S
Institute of Physics, Graduate School of Arts and Sciences, University of Tokyo at Komaba, Japan.
Biochemistry. 1995 Aug 15;34(32):10113-9. doi: 10.1021/bi00032a003.
Purified liver microsomal cytochrome P450IA2 or P450IIB4 was co-reconstituted with cytochrome b5 or NADPH-cytochrome P450 reductase in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles at a lipid to P450 weight ratio of 2 by cholate dialysis procedures. The proteoliposomes catalyzed drug oxidation. Rotational diffusion of cytochrome P450 was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The absorption anisotropy decayed within 1 ms to a time-independent value, r3. Different rotational mobility for the two cytochrome P450s was observed. Though 20% of cytochrome P450IA2 was immobile, all cytochrome P450IIB4 molecules were rotating. The rotational relaxation time, phi, of the mobile population was 237 microseconds for cytochrome P450IA2 and 160 microseconds for cytochrome P450IIB4. The two cytochrome P450s have shown very different interactions with cytochrome b5 and NADPH-cytochrome P450 reductase. By the presence of the redox partner, the mobile population of cytochrome P450IA2 was increased significantly from 80% to 96% (plus cytochrome b5) and to 89% (plus NADPH-cytochrome P450 reductase) due to dissociation of P450 oligomers. On the other hand, the mobility of cytochrome P450IIB4 was not considerably affected by the presence of cytochrome b5 or NADPH-cytochrome P450 reductase as judged by little difference in phi and r3, keeping the mobile population of 100%. These results imply that cytochrome P450IA2 forms a transient association with cytochrome b5 and NADPH-cytochrome P450 reductase.(ABSTRACT TRUNCATED AT 250 WORDS)
通过胆酸盐透析程序，将纯化的肝微粒体细胞色素P450IA2或P450IIB4与细胞色素b5或NADPH - 细胞色素P450还原酶在磷脂酰胆碱 - 磷脂酰乙醇胺 - 磷脂酰丝氨酸囊泡中以脂质与P450的重量比为2进行共重组。蛋白脂质体催化药物氧化。通过观察血红素 - CO复合物光解后吸收各向异性r(t)的衰减来测量细胞色素P450的旋转扩散。r(t)的分析基于“绕膜法线旋转”模型。吸收各向异性在1毫秒内衰减至与时间无关的值r3。观察到两种细胞色素P450具有不同的旋转流动性。虽然20%的细胞色素P450IA2是不动的，但所有细胞色素P450IIB4分子都在旋转。细胞色素P450IA2可移动群体的旋转弛豫时间φ为237微秒，细胞色素P450IIB4为160微秒。两种细胞色素P450与细胞色素b5和NADPH - 细胞色素P450还原酶表现出非常不同的相互作用。由于P450寡聚体的解离，通过氧化还原伙伴的存在，细胞色素P450IA2的可移动群体从80%显著增加到96%（加细胞色素b5）和89%（加NADPH - 细胞色素P450还原酶）。另一方面，细胞色素b5或NADPH - 细胞色素P450还原酶的存在对细胞色素P450IIB4的流动性没有显著影响，从φ和r3的微小差异判断，可移动群体保持在100%。这些结果表明细胞色素P450IA2与细胞色素b5和NADPH - 细胞色素P450还原酶形成瞬时缔合。（摘要截短于250字）
