Hernandez-Sotomayor SM, Munoz-Sanchez JA, Loyola-Vargas VM
Unidad de Biologia Experimental, Centro de Investigacion Cientifica de Yucatan, Apartado Postal 87 Cordemex 97310, Merida, Yucatan, Mexico.
Plant Physiol. 1999 Aug;120(4):1075-82. doi: 10.1104/pp.120.4.1075.
The properties of phospholipase C (PLC) partially purified from Catharanthus roseus transformed roots were analyzed using substrate lipids dispersed in phospholipid vesicles, phospholipid-detergent mixed micelles, and phospholipid monolayers spread at an air-water interface. Using [(33)P]phosphatidylinositol 4,5-bisphosphate (PIP(2)) of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphate and its subsequent dissolution on quenching in the subphase. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure. The optimum surface pressure for PIP(2) hydrolysis was 20 mN/m. Depletion of PLC from solution by incubation with sucrose-loaded PIP(2) vesicles followed by ultracentrifugation demonstrated stable attachment of PLC to the vesicles. A mixed micellar system was established to assay PLC activity using deoxycholate. Kinetic analyses were performed to determine whether PLC activity was dependent on both bulk PIP(2) and PIP(2) surface concentrations in the micelles. The interfacial Michaelis constant was calculated to be 0.0518 mol fraction, and the equilibrium dissociation constant of PLC for the lipid was 45.5 &mgr;M. These findings will add to our understanding of the mechanisms of regulation of plant PLC.
利用分散于磷脂囊泡、磷脂 - 去污剂混合胶束以及铺展在空气 - 水界面的磷脂单分子层中的底物脂质，对从长春花转化根中部分纯化得到的磷脂酶C（PLC）的特性进行了分析。使用具有高比放射性的[(33)P]磷脂酰肌醇4,5 - 二磷酸（PIP(2)），通过测量由于肌醇磷酸释放导致单分子层放射性损失以及随后在亚相中猝灭时其溶解情况，直接监测PLC活性。PLC活性受到单分子层表面压力的显著影响，在初始压力极端情况下活性降低。PIP(2)水解的最佳表面压力为20 mN/m。通过与蔗糖负载的PIP(2)囊泡孵育然后超速离心从溶液中去除PLC，证明了PLC与囊泡的稳定附着。建立了一个使用脱氧胆酸盐的混合胶束系统来测定PLC活性。进行动力学分析以确定PLC活性是否依赖于胶束中的本体PIP(2)和PIP(2)表面浓度。计算得出界面米氏常数为0.0518摩尔分数，PLC对脂质的平衡解离常数为45.5 μM。这些发现将增进我们对植物PLC调节机制的理解。
