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Detection of Three Distinct Phospholipases A2 in Cultured Mast Cells1

1992; Oxford University Press; Volume: 111; Issue: 2 Linguagem: Inglês

10.1093/oxfordjournals.jbchem.a123733

1756-2651

Makoto Murakami, Ichiro Kudo, Masato Umeda, Atsushi Matsuzawa, Mayuko Takeda, Masayuki Komada, Yumi Fujimori, Katsuhiko Takahashi, Keizo Inoue,

Biomedical Research and Pathophysiology

Phospholipase A2 activity in lysates of mast cells such as rat mastocytoma RBL-2H3 cells and mouse bone marrow-derived IL-3-dependent mast cells (BMMC) was measured using phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylserine (PS) as a substrate. Both types of cells exhibited phospholipase A2 activity with a similar pH profile; the optimum pH observed with PS as a substrate was 5.5–7.4, whereas that with PE or PC was 8.0–9.0. PE and PC bearing an arachidonate at the sn-2 position were cleaved more efficiently by PE, PC-hydrolyzing phospholipase A2 than phospholipids with a linoleate. A monoclonal antibody raised against rabbit platelet 85-kDa cytosolic phospholipase A2 absorbed the PE, PC-hydrolyzing activity. PS-hydrolyzing activity was purified from RBL-2H3 cells and BMMC by sequential heparin-Sepharose, butyl-Toyo-pearl, and reverse-phase HPLC. On reverse-phase HPLC, the PS-hydrolyzing activity of RBL cells was separated into two peaks, A and B. The peak B activity was inhibited by the anti-rat 14-kDa group II phospholipase A2 antibody, while the peak A activity was not. The partially purified peak A activity hydrolyzed PS about 10-fold more efficiently than PE at optimum pH of 5.5–7.4. No appreciable hydrolysis was observed with PC or phosphatidyl-inositol (PI). Thus, mast cells may express at least three distinct phospholipases A2; 14-kDa group II phospholipase A2, 85-kDa cytosolic arachidonate preferential phospholipase A2 and a novel phospholipase A2 that shows high substrate specificity for PS.