Rebecchi M J, Rosen O M
J Biol Chem. 1987 Sep 15;262(26):12526-32.
A soluble phosphoinositide-specific phospholipase C (PLC) was purified 58,000-fold from bovine brain. The enzyme, one of six distinct PLC activities detected in brain, accounted for approximately 15% of the soluble phosphatidylinositol-4,5-bisphosphate-phospholipase C (PIP2-PLC) activity in this tissue. The purification scheme included hydrophobic chromatography on phenyl-Sepharose and affinity chromatography on phosphatidylinositol-Sepharose (PI-Sepharose). The enzyme was specifically eluted from the PI-Sepharose with PI, calcium, and detergent. The purified PLC had an estimated molecular weight of 88,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaved as a monomeric protein during sedimentation on glycerol gradients. The enzyme required calcium for activity, exhibited a pH optimum of 6.5, and cleaved only phosphoinositides. The rates of PIP2 and phosphatidyl-4-monophosphate hydrolysis exceeded the rate of PI hydrolysis under all conditions tested. These properties are consistent with a potential role for this PLC in the early events involved in cellular calcium mobilization.
一种可溶性磷酸肌醇特异性磷脂酶C（PLC）从牛脑中纯化出来，纯化倍数达58000倍。该酶是在脑中检测到的六种不同PLC活性之一，占该组织中可溶性磷脂酰肌醇-4,5-二磷酸磷脂酶C（PIP2-PLC）活性的约15%。纯化方案包括在苯基琼脂糖上进行疏水层析以及在磷脂酰肌醇琼脂糖（PI-琼脂糖）上进行亲和层析。该酶用PI、钙和去污剂从PI-琼脂糖上特异性洗脱。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上，纯化的PLC估计分子量为88000，在甘油梯度沉降过程中表现为单体蛋白。该酶的活性需要钙，最适pH为6.5，且只裂解磷酸肌醇。在所有测试条件下，PIP2和磷脂酰-4-单磷酸的水解速率超过PI的水解速率。这些特性与该PLC在细胞钙动员早期事件中的潜在作用一致。
