James S R, Paterson A, Harden T K, Downes C P
Department of Biochemistry, University of Dundee, Tayside, Scotland, United Kingdom.
J Biol Chem. 1995 May 19;270(20):11872-81. doi: 10.1074/jbc.270.20.11872.
Phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5)-P2) hydrolysis by three different beta-isoforms of phospholipase C (PLC) was examined to investigate the catalytic action of these extracellular signal-regulated enzymes. Depletion of phospholipase C from solution by incubation with sucrose-loaded vesicles of differing compositions followed by ultracentrifugation demonstrated stable attachment of PLC to the vesicles from which an equilibrium association constant of PLC with PtdIns (4,5)P2 could be determined. A mixed micellar system was established to assay PLC activity using dodecyl maltoside, which behaved as an essentially inert diluent of PtdIns (4,5)P2 with respect to PLC beta activity. Kinetic analyses were performed to test whether PLC beta activity was dependent on both bulk PtdIns (4,5)P2 concentration and surface concentration in the micelles as has been shown for other lipid metabolising enzymes. Each of the PLC beta isoforms behaved similarly in these analyses, which indicated the involvement of at least two binding events. Interfacial Michaelis constants were calculated to be between 0.1-0.2 mol fraction for all three enzymes, and Ks (the equilibrium dissociation constant of PLC for lipid) ranged between 100-200 microM. The apparent multiple interfacial binding events did not appear to result from lipid-induced PLC beta oligomerization implying that PLC beta monomers possess more than one lipid-binding site. Surface dilution of PLC-catalyzed PtdIns (4,5)P2 hydrolysis was assessed in the presence of increasing concentrations of various nonsubstrate phospholipids, which profoundly reduced PLC activity, suggesting that these lipids may inhibit enzyme action. The data indicate that G protein-regulated isoforms of PLC operate with separate lipid binding and catalytic steps and imply that under physiological conditions, PLC beta isoforms operate under first-order conditions. These findings may have implications for the mechanisms of regulation of PLC beta s by G protein subunits.
通过三种不同的磷脂酶C（PLC）β同工型对磷脂酰肌醇4,5 - 二磷酸（PtdIns(4,5)-P2）的水解作用进行了研究，以探究这些细胞外信号调节酶的催化作用。通过与不同组成的蔗糖负载囊泡孵育，随后进行超速离心，从溶液中耗尽磷脂酶C，结果表明PLC稳定附着于囊泡，据此可确定PLC与PtdIns(4,5)P2的平衡缔合常数。建立了一个混合胶束系统，使用十二烷基麦芽糖苷来测定PLC活性，对于PLCβ活性而言，十二烷基麦芽糖苷表现为PtdIns(4,5)P2的基本惰性稀释剂。进行了动力学分析，以测试PLCβ活性是否如其他脂质代谢酶那样，既依赖于胶束中PtdIns(4,5)P2的总体浓度，也依赖于其表面浓度。在这些分析中，每种PLCβ同工型表现相似，这表明至少涉及两个结合事件。计算出所有三种酶的界面米氏常数在0.1 - 0.2摩尔分数之间，Ks（PLC对脂质的平衡解离常数）在100 - 200微摩尔之间。明显的多个界面结合事件似乎并非由脂质诱导的PLCβ寡聚化导致，这意味着PLCβ单体拥有不止一个脂质结合位点。在存在浓度不断增加的各种非底物磷脂的情况下，评估了PLC催化的PtdIns(4,5)P2水解的表面稀释情况，结果表明这些磷脂显著降低了PLC活性，提示它们可能抑制酶的作用。数据表明，G蛋白调节的PLC同工型通过独立的脂质结合和催化步骤发挥作用，这意味着在生理条件下，PLCβ同工型在一级条件下发挥作用。这些发现可能对G蛋白亚基调节PLCβ的机制具有启示意义。
