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.
A phospholipase A2 hydrolyzing arachidonic-acid-containing phospholipids has been purified 5600-fold from mouse spleen and to near homogeneity from the macrophage cell line J774. A molecular mass of 100 kDa for the enzyme was estimated by SDS/PAGE, while it migrated as a 70-kDa protein upon gel chromatography. The enzyme from both sources showed the same characteristics as that previously identified in murine peritoneal macrophages [Wijkander, J. & Sundler, R. (1989), FEBS Lett. 244, 51-56], i.e. it was totally dependent on Ca2+ with half-maximal activity at approximately 0.7 microM and hydrolyzed arachidonoyl phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol equally well. Also, the platelet-activating-factor precursor, 1-O-alkyl-2-arachidonoylglycerophosphocholine, was hydrolyzed to a similar extent. A preference for arachidonoylphosphatidylcholine over oleoylphosphatidylcholine was seen both with sonicated vesicles and labeled macrophage membranes as substrate. Ca(2+)-dependent interaction of the enzyme with sonicated vesicles composed of neutral phospholipids led to rapid initial hydrolysis, followed by loss of catalytic activity. Such inactivation did not occur with vesicles of pure anionic phospholipids, or with membranes prepared from macrophages. Phospholipase A2, purified from J774 cells, was rapidly phosphorylated by protein kinase C type-II, leading to incorporation of approximately 0.5 mol phosphate/mol enzyme.
Wijkander
J
J
Department of Medical and Physiological Chemistry, Lund University, Sweden.
Sundler
R
R
D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism*; D002460:Cell Line; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003600:Cytosol / Q000201:enzymology; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006882:Hydroxyapatites; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D010741:Phospholipases A / Q000302:isolation & purification* / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology*; D013379:Substrate Specificity
D001095:Arachidonic Acids; D006882:Hydroxyapatites; D010743:Phospholipids; D017886:Durapatite; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2