Munnik T, de Vrije T, Irvine R F, Musgrave A
Institute for Molecular Cell Biology, BioCentrum Amsterdam, University of Amsterdam, Kruislaan 318, NL-1098 SM, Amsterdam, The Netherlands.
J Biol Chem. 1996 Jun 28;271(26):15708-15. doi: 10.1074/jbc.271.26.15708.
We provide evidence that phosphatidic acid (PtdOH) formed during signaling in plants is metabolized by a novel pathway. In much of this study, 32Pi-labeled Chlamydomonas cells were used, and signaling was activated by adding the G-protein activator mastoparan. Within seconds of activation, large amounts of [32P]PtdOH were formed, with peak production at about 4 min, when the level was 5-25-fold higher than the control. As the level of [32P]PtdOH subsequently decreased, an unknown phospholipid (PLX) increased in radiolabeling; before activation it was barely detectable. The chromatographic properties of PLX resembled those of lyso-PtdOH and CMP.PtdOH but on close inspection were found to be different. PLX was shown to be diacylglycerol pyrophosphate (DGPP), the product of a newly discovered enzyme, phosphatidate kinase, whose in vitro activity was described recently (Wissing, J. B., and Behrbohm, H. (1993) Plant Physiol. 102, 1243-1249). The identity of DGPP was established by co-chromatrography with a standard and by degradation analysis as follows: [32P]DGPP was deacylated, and the product (glycerolpyrophosphate, GroPP) was hydrolyzed by mild acid treatment or pyrophosphatase to produce GroP and Pi as the only radioactive products. Since DGPP is the pyrophosphate derivative of PtdOH and is formed as the concentration of PtdOH decreases, we assumed that PtdOH was converted in vivo to DGPP. This was confirmed by showing that during a short labeling protocol while the specific radioactivity of DGPP was increasing, the specific radioactivity of the 32Pi derived from DGPP as above was higher than that of [32P]GroP. DGPP was also formed in suspension cultures of tomato and potato cells, and its synthesis was activated by mastoparan. Moreover, it was also found in intact tissues of a number of higher plants, for example, carnation flower petals, vetch roots, leaves of fig-leaved goosefoot, and common persicaria and microspores of rape seed. Our results suggest that DGPP is a common but minor plant lipid that increases in concentration when signaling is activated. Possible functions of DGPP in phospholpase C and D signaling cascades are discussed.
我们提供证据表明，植物信号传导过程中形成的磷脂酸（PtdOH）通过一条新途径进行代谢。在本研究的大部分实验中，使用了经32Pi标记的衣藻细胞，并通过添加G蛋白激活剂mastoparan来激活信号传导。激活后数秒内，大量[32P]PtdOH形成，约4分钟时产量达到峰值，此时其水平比对照高5 - 25倍。随着[32P]PtdOH水平随后下降，一种未知磷脂（PLX）的放射性标记增加；激活前几乎检测不到。PLX的色谱特性类似于溶血磷脂酸（lyso - PtdOH）和CMP.PtdOH，但仔细检查发现有所不同。结果表明PLX是二酰基甘油焦磷酸（DGPP），是一种新发现的酶——磷脂酸激酶的产物，其体外活性最近已有描述（Wissing, J. B., and Behrbohm, H. (1993) Plant Physiol. 102, 1243 - 1249）。通过与标准品共色谱分析以及如下降解分析确定了DGPP的身份：[32P]DGPP进行脱酰基反应，产物（甘油焦磷酸，GroPP）经温和酸处理或焦磷酸酶水解后，产生GroP和Pi作为唯一放射性产物。由于DGPP是PtdOH的焦磷酸衍生物，且在PtdOH浓度降低时形成，我们推测PtdOH在体内转化为DGPP。这一点通过以下实验得到证实：在短时间标记实验中，当DGPP的比放射性增加时，上述来自DGPP的32Pi的比放射性高于[32P]GroP的比放射性。番茄和马铃薯细胞的悬浮培养物中也形成DGPP，其合成可被mastoparan激活。此外，在多种高等植物的完整组织中也发现了DGPP，例如康乃馨花瓣、野豌豆根、藜叶、虎杖以及油菜籽小孢子。我们的结果表明，DGPP是一种常见但含量较少的植物脂质，在信号传导激活时浓度会增加。文中还讨论了DGPP在磷脂酶C和D信号级联反应中的可能功能。
