Kusunose E, Ogita K, Ichihara K, Kusunose M
J Biochem. 1981 Oct;90(4):1069-76. doi: 10.1093/oxfordjournals.jbchem.a133558.
Cytochrome P-450 was solubilized from kidney cortex microsomes of rabbits treated with 3-methylcholanthrene and partially purified by chromatography on 6-amino-n-hexyl Sepharose 4B and heparin-Sepharose CL-6B columns. Fatty acid omega- and (omega-1)-hydroxylation activity was reconstituted from the partially purified cytochrome P-450 and NADPH-cytochrome c reductase, with phosphatidylethanolamine or phosphatidylcholine. The activity was further stimulated by addition of detergent-solubilized cytochrome b5 from rabbit liver microsomes. Trypsin-solubilized or boiled detergent-solubilized cytochrome b5 had no effect. Among fatty acids tested, caprate, laurate, myristate, and palmitate were the most effective substrates. When caprate and laurate were used as the substrates, the products were the corresponding omega- and (omega-1)-hydroxy fatty acids. The ratio of these products was altered by addition of cytochrome b5. On the other hand, when myristate and palmitate were the substrates, small amounts of unknown polar fatty acids were also formed besides omega- and (omega-1)-hydroxy fatty acids, and the ratio of these products was not affected by addition of cytochrome b5. Benzo(a)pyrene hydroxylation activity was also reconstituted from the same cytochrome P-450 preparation, NADPH-cytochrome c reductase, and phosphatidylserine. However, cytochrome b5 showed only a slight stimulation. The possibility that different cytochrome P-450 species are involved in fatty acid and benzo(a)pyrene hydroxylations is discussed.
细胞色素P-450从经3-甲基胆蒽处理的兔肾皮质微粒体中溶解出来，并通过在6-氨基正己基琼脂糖4B和肝素-琼脂糖CL-6B柱上进行色谱法部分纯化。脂肪酸ω-和（ω-1）-羟基化活性由部分纯化的细胞色素P-450和NADPH-细胞色素c还原酶与磷脂酰乙醇胺或磷脂酰胆碱一起重建。通过添加来自兔肝微粒体的去污剂溶解的细胞色素b5，该活性进一步受到刺激。胰蛋白酶溶解的或煮沸的去污剂溶解的细胞色素b5没有作用。在所测试的脂肪酸中，癸酸、月桂酸、肉豆蔻酸和棕榈酸是最有效的底物。当使用癸酸和月桂酸作为底物时，产物是相应的ω-和（ω-1）-羟基脂肪酸。这些产物的比例通过添加细胞色素b5而改变。另一方面，当肉豆蔻酸和棕榈酸作为底物时，除了ω-和（ω-1）-羟基脂肪酸外，还形成少量未知的极性脂肪酸，并且这些产物的比例不受添加细胞色素b5的影响。苯并（a）芘羟基化活性也由相同的细胞色素P-450制剂、NADPH-细胞色素c还原酶和磷脂酰丝氨酸重建。然而，细胞色素b5仅显示出轻微的刺激作用。讨论了不同细胞色素P-450种类参与脂肪酸和苯并（a）芘羟基化的可能性。
