Takenawa T, Egawa K
J Biol Chem. 1977 Aug 10;252(15):5419-23.
CDP-diglyceride:inositol transferase, which catalyzes the final step of the de novo synthesis of phosphatidylinositol, was solubilized by sodium cholate from microsomes prepared from rat liver and purified by ammonium sulfate fractionation, sucrose density gradient centrifugation, and DEAE-cellulose column chromatography. Addition of phospholipid during the purification and the assay procedures prevented irreversible loss of the enzyme activity to some extent. The resulting preparation was nearly homogeneous as judged by polyacrylamide gel electrophoresis. The recovery of the purified enzyme from the microsomal fraction was 3 to 3.3% with respect to activity and 0.12% with respect to amount of protein. The molecular weight of the enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 60,000. The purified enzyme required exogenous phospholipds for its activity. Various phospholipid classes activated the enzyme rather nonspecifically. The Km for myo-inositol was 2.5 X 10(-3) M and that for CDP-diglyceride was 1.7 X 10(-4) M. The pH optimum was 8.6. The enzyme required Mm2+ or Mg2+ for activity. The optimal concentration of Mn2+ for activation was 0.5 mM, while the activity in the presence of Mg2+ increased up to 20 mM. The enzyme was inhibited by thiol-reactive reagents. There was a competition for inositol by inosose-2 but not by scyllitol.
催化磷脂酰肌醇从头合成最后一步反应的CDP - 二甘油酯：肌醇转移酶，用胆酸钠从大鼠肝脏制备的微粒体中溶解出来，并通过硫酸铵分级分离、蔗糖密度梯度离心和DEAE - 纤维素柱色谱法进行纯化。在纯化和测定过程中添加磷脂在一定程度上防止了酶活性的不可逆损失。通过聚丙烯酰胺凝胶电泳判断，所得制剂几乎是纯的。从微粒体部分纯化得到的酶，活性回收率为3%至3.3%，蛋白质含量回收率为0.12%。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳估计该酶的分子量为60,000。纯化后的酶活性需要外源性磷脂。各种磷脂类对该酶的激活作用相当非特异性。肌醇的Km值为2.5×10⁻³ M，CDP - 二甘油酯的Km值为1.7×10⁻⁴ M。最适pH为8.6。该酶活性需要Mn²⁺或Mg²⁺。激活的最佳Mn²⁺浓度为0.5 mM，而在Mg²⁺存在下，活性在高达20 mM时增加。该酶被硫醇反应性试剂抑制。肌醇 - 2对肌醇有竞争作用，而半乳糖醇则没有。
