Koop D R, Morgan E T, Tarr G E, Coon M J
J Biol Chem. 1982 Jul 25;257(14):8472-80.
A new isozyme of cytochrome P-450 has been purified to electrophoretic homogeneity from hepatic microsomes of rabbits treated chronically with ethanol. Several criteria indicate that the ethanol-inducible cytochrome, which has a minimal molecular weight of 51,000 and is designated form 3a on the basis of its relative electrophoretic mobility, is distinct from the known isozymes of P-450. As judged spectrally, the new isozyme is high spin in the oxidized state, as is form 4, but differs in that the spin state is unperturbed by nonionic detergents. The absolute spectrum of the ferrous carbonyl complex of form 3a is red shifted as compared to that of forms 2, 3b, 3c, 4, and 6 and exhibits a maximum at 452 nm. The amino acid composition of form 3a is different from that of the other isozymes, and both the NH2- and COOH-terminal sequences are distinct; form 3a has an NH2-terminal alanine and a carboxyl-terminal leucine residue. Peptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following treatment with papain, chymotrypsin, or Staphylococcus aureus V8 protease and by high performance liquid chromatography following trypsinolysis indicates that form 3a is a unique gene product. This cytochrome displays the highest activity of all of the rabbit isozymes in the oxidation of ethanol to acetaldehyde and the p-hydroxylation of aniline when reconstituted with NADPH-cytochrome P-450 reductase and phospholipid in the presence of NADPH and oxygen.
从长期用乙醇处理的兔肝脏微粒体中已将一种新的细胞色素P - 450同工酶纯化至电泳纯。多项标准表明，这种乙醇诱导的细胞色素，其最小分子量为51,000，根据其相对电泳迁移率被命名为3a型，与已知的P - 450同工酶不同。从光谱判断，新的同工酶在氧化态下是高自旋的，与4型相同，但不同之处在于其自旋状态不受非离子去污剂的影响。与2型、3b型、3c型、4型和6型相比，3a型亚铁羰基配合物的绝对光谱发生红移，在452 nm处有一个最大值。3a型的氨基酸组成与其他同工酶不同，其氨基末端和羧基末端序列都不同；3a型有一个氨基末端丙氨酸和一个羧基末端亮氨酸残基。用木瓜蛋白酶、胰凝乳蛋白酶或金黄色葡萄球菌V8蛋白酶处理后通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳进行肽图分析，以及胰蛋白酶消化后通过高效液相色谱分析表明，3a型是一种独特的基因产物。当在NADPH和氧气存在下与NADPH - 细胞色素P - 450还原酶和磷脂重组时，这种细胞色素在将乙醇氧化为乙醛以及苯胺的对羟基化反应中表现出所有兔同工酶中最高的活性。
