Wu W I, Liu Y, Riedel B, Wissing J B, Fischl A S, Carman G M
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick 08903, USA.
J Biol Chem. 1996 Jan 26;271(4):1868-76. doi: 10.1074/jbc.271.4.1868.
Diacylglycerol pyrophosphate (DGPP) phosphatase is a novel membrane-associated enzyme that catalyzes the dephosphorylation of the beta phosphate of DGPP to yield phosphatidate and Pi. DGPP phosphatase was purified 33,333-fold from Saccharomyces cerevisiae by a procedure that included Triton X-100 solubilization of microsomal membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, and Mono Q. The procedure resulted in the isolation of an apparent homogeneous protein with a subunit molecular mass of 34 kDa. DGPP phosphatase activity was associated with the 34-kDa protein. DGPP phosphatase had a broad pH optimum between 6.0 and 8.5 and was dependent on Triton X-100 for maximum activity. The enzyme was inhibited by divalent cations, NaF, and pyrophosphate and was relatively insensitive to thioreactive agents. The turnover number (molecular activity) for the enzyme was 5.8 x 10(3) min-1 at pH 6.5 and 30 degrees C. DGPP phosphatase exhibited typical saturation kinetics with respect to DGPP (Km = 0.55 mol %). The Km value for DGPP was 3-fold greater than its cellular concentration (0.18 mol %). DGPP phosphatase also catalyzed the dephosphorylation of phosphatidate, but this dephosphorylation was subsequent to the dephosphorylation of the beta phosphate of DGPP. The dependence of activity on phosphatidate (Km = 2.2 mol %) was cooperative (Hill number = 2.0). DGPP was the preferred substrate for the enzyme with a specificity constant (Vmax/Km) 10-fold greater than that for phosphatidate. In addition, DGPP potently inhibited (Ki = 0.35 mol %) the dephosphorylation of phosphatidate by a competitive mechanism whereas phosphatidate did not inhibit the dephosphorylation of DGPP. DGPP was neither a substrate nor an inhibitor of pure phosphatidate phosphatase from S. cerevisiae. DGPP was synthesized from phosphatidate via the phosphatidate kinase reaction.
二酰甘油焦磷酸（DGPP）磷酸酶是一种新型的膜相关酶，它催化DGPP的β磷酸去磷酸化，生成磷脂酸和无机磷酸（Pi）。通过一种包括用Triton X-100溶解微粒体膜，然后依次用DE53、Affi-Gel Blue、羟基磷灰石和Mono Q进行层析的方法，从酿酒酵母中纯化出了33333倍的DGPP磷酸酶。该方法得到了一种表观均一的蛋白质，其亚基分子量为34 kDa。DGPP磷酸酶活性与34 kDa的蛋白质相关。DGPP磷酸酶在pH 6.0至8.5之间具有较宽的最适pH值，并且其最大活性依赖于Triton X-100。该酶受到二价阳离子、氟化钠和焦磷酸的抑制，对硫反应性试剂相对不敏感。在pH 6.5和30℃时，该酶的转换数（分子活性）为5.8×10³ min⁻¹。DGPP磷酸酶对DGPP表现出典型的饱和动力学（Km = 0.55 mol%）。DGPP的Km值比其细胞内浓度（0.18 mol%）大3倍。DGPP磷酸酶也催化磷脂酸的去磷酸化，但这种去磷酸化发生在DGPP的β磷酸去磷酸化之后。其活性对磷脂酸的依赖性（Km = 2.2 mol%）具有协同性（希尔系数 = 2.0）。DGPP是该酶的首选底物，其特异性常数（Vmax/Km）比磷脂酸大10倍。此外，DGPP通过竞争性机制强烈抑制（Ki = 0.35 mol%）磷脂酸的去磷酸化，而磷脂酸不抑制DGPP的去磷酸化。DGPP既不是酿酒酵母纯磷脂酸磷酸酶的底物，也不是其抑制剂。DGPP是通过磷脂酸激酶反应由磷脂酸合成的。
