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. 1993 Aug 15;294(Pt 1):261–270. doi: 10.1042/bj2940261

Purification of a 100 kDa phospholipase A2 from spleen, lung and kidney: antiserum raised to pig spleen phospholipase A2 recognizes a similar form in bovine lung, kidney and platelets, and immunoprecipitates phospholipase A2 activity.

D K Kim 1, J V Bonventre 1
PMCID: PMC1134593  PMID: 8363579

Abstract

Phospholipase A2 (PLA2) plays a key role in the production of intracellular and extracellular chemical mediators such as arachidonic acid, eicosanoids and platelet-activating factor, which modulate membrane channel activity, signal transduction, are vasoactive and chemotactic, and are implicated in many pathophysiological mechanisms of inflammation and tissue injury. We previously identified, purified and characterized an arachidonic acid-selective cytosolic 100-110 kDa PLA2 from bovine platelets and rat kidney that is activated during cell stimulation. The purification schemes previously published resulted in low yields of enzyme, insufficient for extensive biochemical characterization. We report the purification of a large-molecular-mass (100 kDa) PLA2 from pig spleen, bovine kidney and bovine lung, using a novel large-scale purification scheme. The enzyme was purified to near homogeneity from an acidified extract obtained from 4.8 kg of pig spleen by sequential use of DEAE-cellulose anionic exchange, Butyl-Toyopearl hydrophobic chromatography and DEAE-5PW h.p.l.c., and further purified by non-denaturing PAGE. This purification scheme will permit the preparation of quantities of purified native enzyme sufficient to study its properties and regulation. To generate antiserum against the PLA2 enzyme, the 100 kDa protein was excised and electroeluted from SDS/PAGE gels of the active fractions after DEAE-5PW h.p.l.c., and this was used as antigen. This polyclonal antibody against pig spleen 100 kDa PLA2 protein reacted with 100 kDa bands in preparations partially purified from bovine platelets, kidney and lung as well as pig spleen, and immunoprecipitated PLA2 activity from these sources. The antibody also immunoprecipitated a 100 kDa protein from cytosolic fractions of cultured renal mesangial cells, human erythroleukaemia cells and human monocytic U937 cells. Considerable PLA2 activity was present in the immunoprecipitates. To our knowledge this antibody is unique in its ability to permit measurement of PLA2 activity in the immunoprecipitate itself, and will be a useful tool for the study of the regulation and the activation mechanisms of the native PLA2 enzyme.

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Selected References

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  1. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  2. Bennett C. F., McCarte A., Crooke S. T. Purification and characterization of a soluble phospholipase A2 from guinea pig lung. Biochim Biophys Acta. 1990 Dec 4;1047(3):271–283. doi: 10.1016/0005-2760(90)90526-4. [DOI] [PubMed] [Google Scholar]
  3. Bonventre J. V., Gronich J. H., Nemenoff R. A. Epidermal growth factor enhances glomerular mesangial cell soluble phospholipase A2 activity. J Biol Chem. 1990 Mar 25;265(9):4934–4938. [PubMed] [Google Scholar]
  4. Bonventre J. V., Nemenoff R. Renal tubular arachidonic acid metabolism. Kidney Int. 1991 Mar;39(3):438–449. doi: 10.1038/ki.1991.55. [DOI] [PubMed] [Google Scholar]
  5. Bonventre J. V. Phospholipase A2 and signal transduction. J Am Soc Nephrol. 1992 Aug;3(2):128–150. doi: 10.1681/ASN.V32128. [DOI] [PubMed] [Google Scholar]
  6. Bonventre J. V., Skorecki K. L., Kreisberg J. I., Cheung J. Y. Vasopressin increases cytosolic free calcium concentration in glomerular mesangial cells. Am J Physiol. 1986 Jul;251(1 Pt 2):F94–102. doi: 10.1152/ajprenal.1986.251.1.F94. [DOI] [PubMed] [Google Scholar]
  7. Channon J. Y., Leslie C. C. A calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase A2 with membrane in the macrophage cell line RAW 264.7. J Biol Chem. 1990 Apr 5;265(10):5409–5413. [PubMed] [Google Scholar]
  8. Clark J. D., Lin L. L., Kriz R. W., Ramesha C. S., Sultzman L. A., Lin A. Y., Milona N., Knopf J. L. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP. Cell. 1991 Jun 14;65(6):1043–1051. doi: 10.1016/0092-8674(91)90556-e. [DOI] [PubMed] [Google Scholar]
  9. Clark J. D., Milona N., Knopf J. L. Purification of a 110-kilodalton cytosolic phospholipase A2 from the human monocytic cell line U937. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7708–7712. doi: 10.1073/pnas.87.19.7708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DOLE V. P., MEINERTZ H. Microdetermination of long-chain fatty acids in plasma and tissues. J Biol Chem. 1960 Sep;235:2595–2599. [PubMed] [Google Scholar]
  11. Do S. I., Enns C., Cummings R. D. Human transferrin receptor contains O-linked oligosaccharides. J Biol Chem. 1990 Jan 5;265(1):114–125. [PubMed] [Google Scholar]
  12. Durham A. C. A survey of readily available chelators for buffering calcium ion concentrations in physiological solutions. Cell Calcium. 1983 Feb;4(1):33–46. doi: 10.1016/0143-4160(83)90047-7. [DOI] [PubMed] [Google Scholar]
  13. Edelson J. D., Vadas P., Villar J., Mullen J. B., Pruzanski W. Acute lung injury induced by phospholipase A2. Structural and functional changes. Am Rev Respir Dis. 1991 May;143(5 Pt 1):1102–1109. doi: 10.1164/ajrccm/143.5_Pt_1.1102. [DOI] [PubMed] [Google Scholar]
  14. Filgueiras O. M., Possmayer F. Purification and characterization of a phospholipase A2 associated with rabbit lung microsomes: some evidence for its mitochondrial origin. Biochim Biophys Acta. 1990 Oct 1;1046(3):258–266. doi: 10.1016/0005-2760(90)90239-t. [DOI] [PubMed] [Google Scholar]
  15. Gronich J. H., Bonventre J. V., Nemenoff R. A. Identification and characterization of a hormonally regulated form of phospholipase A2 in rat renal mesangial cells. J Biol Chem. 1988 Nov 15;263(32):16645–16651. [PubMed] [Google Scholar]
  16. Gronich J. H., Bonventre J. V., Nemenoff R. A. Purification of a high-molecular-mass form of phospholipase A2 from rat kidney activated at physiological calcium concentrations. Biochem J. 1990 Oct 1;271(1):37–43. doi: 10.1042/bj2710037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kim D. K., Kudo I., Inoue K. Purification and characterization of rabbit platelet cytosolic phospholipase A2. Biochim Biophys Acta. 1991 Apr 24;1083(1):80–88. doi: 10.1016/0005-2760(91)90127-4. [DOI] [PubMed] [Google Scholar]
  18. Kim D. K., Suh P. G., Ryu S. H. Purification and some properties of a phospholipase A2 from bovine platelets. Biochem Biophys Res Commun. 1991 Jan 15;174(1):189–196. doi: 10.1016/0006-291x(91)90504-z. [DOI] [PubMed] [Google Scholar]
  19. Kramer R. M., Roberts E. F., Manetta J., Putnam J. E. The Ca2(+)-sensitive cytosolic phospholipase A2 is a 100-kDa protein in human monoblast U937 cells. J Biol Chem. 1991 Mar 15;266(8):5268–5272. [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Leslie C. C. Kinetic properties of a high molecular mass arachidonoyl-hydrolyzing phospholipase A2 that exhibits lysophospholipase activity. J Biol Chem. 1991 Jun 15;266(17):11366–11371. [PubMed] [Google Scholar]
  22. Lin L. L., Lin A. Y., Knopf J. L. Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6147–6151. doi: 10.1073/pnas.89.13.6147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lindahl M., von Schenck H., Tagesson C. Isolation and characterization of phospholipase A2 from rat lung with affinity chromatography and two-dimensional gel electrophoresis. Biochim Biophys Acta. 1989 Oct 17;1005(3):282–288. doi: 10.1016/0005-2760(89)90050-7. [DOI] [PubMed] [Google Scholar]
  24. Ono T., Tojo H., Kuramitsu S., Kagamiyama H., Okamoto M. Purification and characterization of a membrane-associated phospholipase A2 from rat spleen. Its comparison with a cytosolic phospholipase A2 S-1. J Biol Chem. 1988 Apr 25;263(12):5732–5738. [PubMed] [Google Scholar]
  25. Parekh R. B., Tse A. G., Dwek R. A., Williams A. F., Rademacher T. W. Tissue-specific N-glycosylation, site-specific oligosaccharide patterns and lentil lectin recognition of rat Thy-1. EMBO J. 1987 May;6(5):1233–1244. doi: 10.1002/j.1460-2075.1987.tb02359.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pierik A. J., Nijssen J. G., Aarsman A. J., Van den Bosch H. Calcium-independent phospholipase A2 in rat tissue cytosols. Biochim Biophys Acta. 1988 Oct 14;962(3):345–353. doi: 10.1016/0005-2760(88)90264-0. [DOI] [PubMed] [Google Scholar]
  27. Rademacher T. W., Parekh R. B., Dwek R. A. Glycobiology. Annu Rev Biochem. 1988;57:785–838. doi: 10.1146/annurev.bi.57.070188.004033. [DOI] [PubMed] [Google Scholar]
  28. Schalkwijk C. G., Märki F., Van den Bosch H. Studies on the acyl-chain selectivity of cellular phospholipases A2. Biochim Biophys Acta. 1990 May 1;1044(1):139–146. doi: 10.1016/0005-2760(90)90229-q. [DOI] [PubMed] [Google Scholar]
  29. Sharp J. D., White D. L., Chiou X. G., Goodson T., Gamboa G. C., McClure D., Burgett S., Hoskins J., Skatrud P. L., Sportsman J. R. Molecular cloning and expression of human Ca(2+)-sensitive cytosolic phospholipase A2. J Biol Chem. 1991 Aug 15;266(23):14850–14853. [PubMed] [Google Scholar]
  30. Takamura H., Narita H., Park H. J., Tanaka K., Matsuura T., Kito M. Differential hydrolysis of phospholipid molecular species during activation of human platelets with thrombin and collagen. J Biol Chem. 1987 Feb 15;262(5):2262–2269. [PubMed] [Google Scholar]
  31. Tojo H., Ono T., Kuramitsu S., Kagamiyama H., Okamoto M. A phospholipase A2 in the supernatant fraction of rat spleen. Its similarity to rat pancreatic phospholipase A2. J Biol Chem. 1988 Apr 25;263(12):5724–5731. [PubMed] [Google Scholar]
  32. Vadas P., Pruzanski W. Role of secretory phospholipases A2 in the pathobiology of disease. Lab Invest. 1986 Oct;55(4):391–404. [PubMed] [Google Scholar]
  33. Wijkander J., Sundler R. An 100-kDa arachidonate-mobilizing phospholipase A2 in mouse spleen and the macrophage cell line J774. Purification, substrate interaction and phosphorylation by protein kinase C. Eur J Biochem. 1991 Dec 18;202(3):873–880. doi: 10.1111/j.1432-1033.1991.tb16445.x. [DOI] [PubMed] [Google Scholar]

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