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. 1998 Dec 1;336(Pt 2):483–489. doi: 10.1042/bj3360483

Purification and characterization of a lysophosphatidic acid-specific phosphatase.

M Hiroyama 1, T Takenawa 1
PMCID: PMC1219894  PMID: 9820827

Abstract

Lysophosphatidic acid (LPA)-specific phosphatase was purified 3300-fold from bovine brain cytosol. The purification was achieved by (NH4)2SO4 fractionation and several chromatography steps, such as Q-Sepharose, DEAE-5PW, Superdex 200 and heparin-Sepharose. The final enzyme preparation showed a single band of molecular mass 44 kDa on SDS/PAGE under reducing conditions. The enzyme activity was completely dependent on the presence of detergents such as Triton X-100, CHAPS, cholate and octyl-beta-glucoside. The activity was independent of Mg2+; other cations were inhibitory. The enzyme hydrolysed LPA specifically but not cardiolipin, tetraoleoyl-bisphosphatidic acid, ceramide 1-phosphate or sphingosine 1-phosphate, although phosphatidic acid was hydrolysed slightly. The purified enzyme hydrolysed 1-oleoyl LPA at a rate of 1. 1 micromol/min per mg of protein when assayed with LPA as Triton X-100 mixed micelles. The Km value for LPA was 38 microM. NaF and N-ethylmaleimide markedly inhibited the activity, but propranolol had a less potent inhibitory effect. The LPA-specific phosphatase might have an important role in LPA elimination.

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Selected References

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  1. Balboa M. A., Balsinde J., Jones S. S., Dennis E. A. Identity between the Ca2+-independent phospholipase A2 enzymes from P388D1 macrophages and Chinese hamster ovary cells. J Biol Chem. 1997 Mar 28;272(13):8576–8580. doi: 10.1074/jbc.272.13.8576. [DOI] [PubMed] [Google Scholar]
  2. Bell R. M., Coleman R. A. Enzymes of glycerolipid synthesis in eukaryotes. Annu Rev Biochem. 1980;49:459–487. doi: 10.1146/annurev.bi.49.070180.002331. [DOI] [PubMed] [Google Scholar]
  3. Bishop W. R., Bell R. M. Assembly of phospholipids into cellular membranes: biosynthesis, transmembrane movement and intracellular translocation. Annu Rev Cell Biol. 1988;4:579–610. doi: 10.1146/annurev.cb.04.110188.003051. [DOI] [PubMed] [Google Scholar]
  4. Daae L. N. The acylation of glycerol 3 -phosphate in different rat organs and in the liver of different species (including man). Biochim Biophys Acta. 1973 May 24;306(2):186–193. doi: 10.1016/0005-2760(73)90224-5. [DOI] [PubMed] [Google Scholar]
  5. Eberhardt C., Gray P. W., Tjoelker L. W. Human lysophosphatidic acid acyltransferase. cDNA cloning, expression, and localization to chromosome 9q34.3. J Biol Chem. 1997 Aug 8;272(32):20299–20305. doi: 10.1074/jbc.272.32.20299. [DOI] [PubMed] [Google Scholar]
  6. Gaits F., Fourcade O., Le Balle F., Gueguen G., Gaigé B., Gassama-Diagne A., Fauvel J., Salles J. P., Mauco G., Simon M. F. Lysophosphatidic acid as a phospholipid mediator: pathways of synthesis. FEBS Lett. 1997 Jun 23;410(1):54–58. doi: 10.1016/s0014-5793(97)00411-0. [DOI] [PubMed] [Google Scholar]
  7. Gomez-Muñoz A., Hatch G. M., Martin A., Jamal Z., Vance D. E., Brindley D. N. Effects of okadaic acid on the activities of two distinct phosphatidate phosphohydrolases in rat hepatocytes. FEBS Lett. 1992 Apr 13;301(1):103–106. doi: 10.1016/0014-5793(92)80219-7. [DOI] [PubMed] [Google Scholar]
  8. Guo Z., Liliom K., Fischer D. J., Bathurst I. C., Tomei L. D., Kiefer M. C., Tigyi G. Molecular cloning of a high-affinity receptor for the growth factor-like lipid mediator lysophosphatidic acid from Xenopus oocytes. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14367–14372. doi: 10.1073/pnas.93.25.14367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Haldar D., Kelker H. C., Pullman M. E. Properties of acyl coenzyme A: 1-acyl-sn-glycerol 3-phosphate acyltransferase from rat liver mitochondria and microsomes. Trans N Y Acad Sci. 1983;41:173–182. doi: 10.1111/j.2164-0947.1983.tb02799.x. [DOI] [PubMed] [Google Scholar]
  10. Haldar D., Lipfert L. Export of mitochondrially synthesized lysophosphatidic acid. J Biol Chem. 1990 Jul 5;265(19):11014–11016. [PubMed] [Google Scholar]
  11. Haldar D., Tso W. W., Pullman M. E. The acylation of sn-glycerol 3-phosphate in mammalian organs and Ehrlich ascites tumor cells. J Biol Chem. 1979 Jun 10;254(11):4502–4509. [PubMed] [Google Scholar]
  12. Hecht J. H., Weiner J. A., Post S. R., Chun J. Ventricular zone gene-1 (vzg-1) encodes a lysophosphatidic acid receptor expressed in neurogenic regions of the developing cerebral cortex. J Cell Biol. 1996 Nov;135(4):1071–1083. doi: 10.1083/jcb.135.4.1071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jamal Z., Martin A., Gomez-Muñoz A., Brindley D. N. Plasma membrane fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that in the endoplasmic reticulum and cytosol. J Biol Chem. 1991 Feb 15;266(5):2988–2996. [PubMed] [Google Scholar]
  14. Kai M., Wada I., Imai S., Sakane F., Kanoh H. Identification and cDNA cloning of 35-kDa phosphatidic acid phosphatase (type 2) bound to plasma membranes. Polymerase chain reaction amplification of mouse H2O2-inducible hic53 clone yielded the cDNA encoding phosphatidic acid phosphatase. J Biol Chem. 1996 Aug 2;271(31):18931–18938. doi: 10.1074/jbc.271.31.18931. [DOI] [PubMed] [Google Scholar]
  15. Kanoh H., Imai S., Yamada K., Sakane F. Purification and properties of phosphatidic acid phosphatase from porcine thymus membranes. J Biol Chem. 1992 Dec 15;267(35):25309–25314. [PubMed] [Google Scholar]
  16. Monroy G., Rola F. H., Pullman M. E. A substrate- and position-specific acylation of sn-glycerol 3-phosphate by rat liver mitochondria. J Biol Chem. 1972 Nov 10;247(21):6884–6894. [PubMed] [Google Scholar]
  17. Moolenaar W. H., Jalink K., van Corven E. J. Lysophosphatidic acid: a bioactive phospholipid with growth factor-like properties. Rev Physiol Biochem Pharmacol. 1992;119:47–65. doi: 10.1007/3540551921_3. [DOI] [PubMed] [Google Scholar]
  18. Moolenaar W. H., Kranenburg O., Postma F. R., Zondag G. C. Lysophosphatidic acid: G-protein signalling and cellular responses. Curr Opin Cell Biol. 1997 Apr;9(2):168–173. doi: 10.1016/s0955-0674(97)80059-2. [DOI] [PubMed] [Google Scholar]
  19. Moolenaar W. H. Lysophosphatidic acid, a multifunctional phospholipid messenger. J Biol Chem. 1995 Jun 2;270(22):12949–12952. doi: 10.1074/jbc.270.22.12949. [DOI] [PubMed] [Google Scholar]
  20. Stern W., Pullman M. E. Acyl-CoA:sn-glycerol-3-phosphate acyltransferase and the positional distribution of fatty acids in phospholipids of cultured cells. J Biol Chem. 1978 Nov 25;253(22):8047–8055. [PubMed] [Google Scholar]
  21. Tang J., Kriz R. W., Wolfman N., Shaffer M., Seehra J., Jones S. S. A novel cytosolic calcium-independent phospholipase A2 contains eight ankyrin motifs. J Biol Chem. 1997 Mar 28;272(13):8567–8575. doi: 10.1074/jbc.272.13.8567. [DOI] [PubMed] [Google Scholar]
  22. Thompson F. J., Clark M. A. Purification of a lysophosphatidic acid-hydrolysing lysophospholipase from rat brain. Biochem J. 1994 Jun 1;300(Pt 2):457–461. doi: 10.1042/bj3000457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Thomson F. J., Clark M. A. Purification of a phosphatidic-acid-hydrolysing phospholipase A2 from rat brain. Biochem J. 1995 Feb 15;306(Pt 1):305–309. doi: 10.1042/bj3060305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Waggoner D. W., Gómez-Muñoz A., Dewald J., Brindley D. N. Phosphatidate phosphohydrolase catalyzes the hydrolysis of ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate. J Biol Chem. 1996 Jul 12;271(28):16506–16509. doi: 10.1074/jbc.271.28.16506. [DOI] [PubMed] [Google Scholar]
  25. Waggoner D. W., Martin A., Dewald J., Gómez-Muñoz A., Brindley D. N. Purification and characterization of novel plasma membrane phosphatidate phosphohydrolase from rat liver. J Biol Chem. 1995 Aug 18;270(33):19422–19429. doi: 10.1074/jbc.270.33.19422. [DOI] [PubMed] [Google Scholar]
  26. Wolf M. J., Gross R. W. Expression, purification, and kinetic characterization of a recombinant 80-kDa intracellular calcium-independent phospholipase A2. J Biol Chem. 1996 Nov 29;271(48):30879–30885. doi: 10.1074/jbc.271.48.30879. [DOI] [PubMed] [Google Scholar]
  27. Xie M., Low M. G. Identification and characterization of an ecto-(lyso)phosphatidic acid phosphatase in PAM212 keratinocytes. Arch Biochem Biophys. 1994 Jul;312(1):254–259. doi: 10.1006/abbi.1994.1307. [DOI] [PubMed] [Google Scholar]

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