Identification of diacylglycerol pyrophosphate as a novel metabolic product of phosphatidic acid during G-protein activation in plants

• Published in: The Journal of biological chemistry Vol. 271; no. 26; pp. 15708 - 15715
• Main Authors: Munnik, T, de Vrije, T, Irvine, R F, Musgrave, A
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 28.06.1996
• Subjects: Agrotechnological Research Institute; Animals; cell culture; Chlamydomonas; Chlamydomonas - metabolism; cultivo de celulas; culture de cellule; Diphosphates - metabolism; fosfolipidos; gliceridos; glyceride; glycerides; GTP-Binding Proteins - metabolism; Instituut voor Agrotechnologisch Onderzoek; Intercellular Signaling Peptides and Proteins; metabolism; metabolisme; metabolismo; Peptides; phosphatide; Phosphatidic Acids - metabolism; Phospholipase D - metabolism; phospholipids; Phospholipids - metabolism; Plants - metabolism; Signal Transduction; technique des traceurs; tecnicas de trazadores; tracer techniques; Type C Phospholipases - metabolism; Wasp Venoms - pharmacology
• ISSN: 0021-9258; 1083-351X
• 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]Pt-dOH 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-chromatography 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.