Ono T, Nakazono K, Kosaka H
Biochim Biophys Acta. 1982 Dec 6;709(1):84-90. doi: 10.1016/0167-4838(82)90424-1.
Squalene epoxidase (EC 1.14.99.7, squalene 2,3-monooxygenase (epoxidizing) was purified to an apparent homogeneity from rat liver microsomes. The purification was carried out by solubilization of microsomes by Triton X-100, fractionation with ion exchangers, hydroxyapatite, Cibacron Blue Sepharose 4B, and chromatofocusing column chromatography. A total purification of 143-fold over the first DEAE-cellulose fraction was achieved. The purified enzyme gave a single major band on SDS-polyacrylamide gel electrophoresis and the Mr was estimated to be 51 000 as a single polypeptide chain. The enzyme showed no distinct absorption spectrum in the visible regions. The squalene epoxidase activity was reconstituted with the purified enzyme, NADPH-cytochrome P-450 reductase (EC 1.6.2.4), FAD, NADPH and molecular oxygen in the presence of Triton X-100. The apparent Michaelis constants for squalene and FAD were 13 microM and 5 microM, respectively. The Vmax was about 186 nmol per mg protein per 30 min for 2,3-oxidosqualene. The enzyme activity was not inhibited by potent inhibitors of cytochrome P-450. It is suggested that squalene epoxidase is distinct from cytochrome P-450 isozymes.
角鲨烯环氧酶（EC 1.14.99.7，角鲨烯2,3-单加氧酶（环氧化））从大鼠肝脏微粒体中纯化至表观均一。纯化过程包括用Triton X-100溶解微粒体，通过离子交换剂、羟基磷灰石、Cibacron Blue Sepharose 4B分级分离以及进行色谱聚焦柱色谱。相对于第一个DEAE-纤维素级分，实现了143倍的总纯化。纯化后的酶在SDS-聚丙烯酰胺凝胶电泳上呈现单一主要条带，作为单条多肽链，其Mr估计为51000。该酶在可见光区域没有明显的吸收光谱。在Triton X-100存在的情况下，用纯化的酶、NADPH-细胞色素P-450还原酶（EC 1.6.2.4）、FAD、NADPH和分子氧重建了角鲨烯环氧酶活性。角鲨烯和FAD的表观米氏常数分别为13μM和5μM。对于2,3-氧化角鲨烯，Vmax约为每毫克蛋白质每30分钟186 nmol。该酶活性不受细胞色素P-450的强效抑制剂抑制。提示角鲨烯环氧酶与细胞色素P-450同工酶不同。
