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. 1987 Aug 1;245(3):893–897. doi: 10.1042/bj2450893

Phosphorylation of partially purified 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen.

J Gomez-Cambronero 1, J M Mato 1, F Vivanco 1, M Sanchez-Crespo 1
PMCID: PMC1148213  PMID: 3663199

Abstract

A new improved method for purification of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) from rat spleen is described. The catalytic subunit of cyclic AMP-dependent protein kinase in the presence of MgATP stimulated about 3-fold the activity of this partially purified enzyme activity. When [gamma-32P]ATP was included in the assay mixture, the analysis of phosphoprotein products by SDS/polyacrylamide-gel electrophoresis and autoradiography showed the incorporation of [32P]phosphate into a single protein band of about 30 kDa. Analysis of the phosphorylated amino acids indicated that the phosphate was incorporated into a serine residue. Activation of the acetylation reaction by the protein kinase was reversible. The reversal of the activation was coincident with the loss of the [32P]phosphate incorporated into the 30 kDa protein band, which suggests that the acetyltransferase is regulated by a phosphorylation-dephosphorylation mechanism dependent on cyclic AMP.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahmad H., Saleemuddin M. A Coomassie blue-binding assay for the microquantitation of immobilized proteins. Anal Biochem. 1985 Aug 1;148(2):533–541. doi: 10.1016/0003-2697(85)90264-7. [DOI] [PubMed] [Google Scholar]
  2. Alonso F., Gil M. G., Sánchez-Crespo M., Mato J. M. Activation of 1-alkyl-2-lysoglycero-3-phosphocholine. Acetyl-CoA transferase during phagocytosis in human polymorphonuclear leukocytes. J Biol Chem. 1982 Apr 10;257(7):3376–3378. [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Domenech C., Machado-De Domenech E., Söling H. D. Regulation of acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine O2-acetyltransferase (lyso-PAF-acetyltransferase) in exocrine glands. Evidence for an activation via phosphorylation by calcium/calmodulin-dependent protein kinase. J Biol Chem. 1987 Apr 25;262(12):5671–5676. [PubMed] [Google Scholar]
  5. Gomez-Cambronero J., Velasco S., Sanchez-Crespo M., Vivanco F., Mato J. M. Partial purification and characterization of 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen. Biochem J. 1986 Jul 15;237(2):439–445. doi: 10.1042/bj2370439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gómez-Cambronero J., Iñarrea P., Alonso F., Sánchez Crespo M. The role of calcium ions in the process of acetyltransferase activation during the formation of platelet-activating factor (PAF-acether). Biochem J. 1984 Apr 15;219(2):419–424. doi: 10.1042/bj2190419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gómez-Cambronero J., Nieto M. L., Mato J. M., Sánchez-Crespo M. Modulation of lyso-platelet-activating factor: acetyl-CoA acetyltransferase from rat splenic microsomes. The role of calcium ions. Biochim Biophys Acta. 1985 Jun 30;845(3):511–515. doi: 10.1016/0167-4889(85)90218-6. [DOI] [PubMed] [Google Scholar]
  8. Gómez-Cambronero J., Velasco S., Mato J. M., Sánchez-Crespo M. Modulation of lyso-platelet activating factor: acetyl-CoA acetyltransferase from rat splenic microsomes. The role of cyclic AMP-dependent protein kinase. Biochim Biophys Acta. 1985 Jun 30;845(3):516–519. doi: 10.1016/0167-4889(85)90219-8. [DOI] [PubMed] [Google Scholar]
  9. Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lee T. C., Malone B., Snyder F. A new de novo pathway for the formation of 1-alkyl-2-acetyl-sn-glycerols, precursors of platelet activating factor. Biochemical characterization of 1-alkyl-2-lyso-sn-glycero-3-P:acetyl-CoA acetyltransferase in rat spleen. J Biol Chem. 1986 Apr 25;261(12):5373–5377. [PubMed] [Google Scholar]
  11. Lenihan D. J., Lee T. C. Regulation of platelet activating factor synthesis: modulation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase by phosphorylation and dephosphorylation in rat spleen microsomes. Biochem Biophys Res Commun. 1984 May 16;120(3):834–839. doi: 10.1016/s0006-291x(84)80182-5. [DOI] [PubMed] [Google Scholar]
  12. Ninio E., Mencia-Huerta J. M., Heymans F., Benveniste J. Biosynthesis of platelet-activating factor. I. Evidence for an acetyl-transferase activity in murine macrophages. Biochim Biophys Acta. 1982 Jan 15;710(1):23–31. doi: 10.1016/0005-2760(82)90185-0. [DOI] [PubMed] [Google Scholar]
  13. Pajares M. A., Villalba M., Mato J. M. Purification of phospholipid methyltransferase from rat liver microsomal fraction. Biochem J. 1986 Aug 1;237(3):699–705. doi: 10.1042/bj2370699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ribbes G., Ninio E., Fontan P., Record M., Chap H., Benveniste J., Douste-Blazy L. Evidence that biosynthesis of platelet-activating factor (paf-acether) by human neutrophils occurs in an intracellular membrane. FEBS Lett. 1985 Oct 28;191(2):195–199. doi: 10.1016/0014-5793(85)80007-7. [DOI] [PubMed] [Google Scholar]
  15. Smolen J. E., Weissmann G. Stimuli which provoke secretion of azurophil enzymes from human neutrophils induce increments in adenosine cyclic 3'-5'-monophosphate. Biochim Biophys Acta. 1981 Jan 21;672(2):197–206. doi: 10.1016/0304-4165(81)90393-7. [DOI] [PubMed] [Google Scholar]
  16. Tateson J. E., Moncada S., Vane J. R. Effects of prostacyclin (PGX) on cyclic AMP concentrations in human platelets. Prostaglandins. 1977 Mar;13(3):389–397. doi: 10.1016/0090-6980(77)90019-3. [DOI] [PubMed] [Google Scholar]
  17. Thomas G., Thomas G., Luther H. Transcriptional and translational control of cytoplasmic proteins after serum stimulation of quiescent Swiss 3T3 cells. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5712–5716. doi: 10.1073/pnas.78.9.5712. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wykle R. L., Malone B., Snyder F. Enzymatic synthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, a hypotensive and platelet-aggregating lipid. J Biol Chem. 1980 Nov 10;255(21):10256–10260. [PubMed] [Google Scholar]
  19. Yeaman S. J., Cohen P., Watson D. C., Dixon G. H. The substrate specificity of adenosine 3':5'-cyclic monophosphate-dependent protein kinase of rabbit skeletal muscle. Biochem J. 1977 Feb 15;162(2):411–421. doi: 10.1042/bj1620411. [DOI] [PMC free article] [PubMed] [Google Scholar]

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