Honjo K, Ishibashi T, Imai Y
J Biochem. 1985 Mar;97(3):955-9. doi: 10.1093/oxfordjournals.jbchem.a135137.
The terminal oxidase of the NADH-dependent lathosterol 5-desaturation system was solubilized from rat liver microsomes with 2% Triton X-100, and partially purified approximately 18-fold with 19% yield after DEAE-cellulose and 6-aminohexyl-Sepharose column chromatography. The final enzyme preparation was free from other electron transfer components and phospholipids in microsomes, and the desaturation reaction was reconstituted with the following components: NADH, molecular oxygen, phospholipids and three proteins, i.e., NADH-cytochrome b5 reductase, cytochrome b5 and the terminal oxidase. Omission of one of these components led to an almost complete loss of the desaturase activity. Under the reconstitution conditions, the desaturase activity was significantly inhibited by potassium cyanide but was not affected by -SH reagents such as N-ethylmaleimide and dithiothreitol.
采用2% Triton X-100从大鼠肝脏微粒体中溶解出NADH依赖的羊毛甾醇5-去饱和系统的末端氧化酶，经DEAE-纤维素和6-氨基己基-琼脂糖柱层析后，该酶得到部分纯化，纯化倍数约为18倍，产率为19%。最终的酶制剂不含微粒体中的其他电子传递成分和磷脂，利用以下成分重建去饱和反应：NADH、分子氧、磷脂和三种蛋白质，即NADH-细胞色素b5还原酶、细胞色素b5和末端氧化酶。缺少其中任何一种成分都会导致去饱和酶活性几乎完全丧失。在重建条件下，去饱和酶活性受到氰化钾的显著抑制，但不受N-乙基马来酰亚胺和二硫苏糖醇等巯基试剂的影响。
