Purification and characterization of a cytosolic, 42-kDa and Ca2+-dependent phospholipase A(2) from bovine red blood cells - Its involvement in Ca2+-dependent release of arachidonic acid from mammalian red blood cells

Abstract

It has become evident that a Ca2+-dependent release of arachidonic acid (AA) and subsequent formation of bioactive lipid mediators such as prostaglandins and leukotrienes in red blood cells (RBCs) can modify physiological functions of neighboring RECs and platelets. Here we identified a novel type of cytosolic PLA(2) in bovine and human RBCs and purified it to apparent homogeneity with a 14,000-fold purification. The purified enzyme, termed rPLA(2), has a molecular mass of 42 kDa and reveals biochemical properties similar to group IV cPLA(2), but shows different profiles from cPLA, in several column chromatographies. Moreover, rPLA2 did not react with any of anti-cPLA(2) and anti-cPLA(2) antibodies and was identified as an unknown protein in matrix-assisted laser desorption/ ionization time-of-flight mass spectrometric analysis. Divalent metal ions tested exhibited similar effects between rPLA(2) and cPLA(2), whereas mercurials inhibited cPLA(2) but had no effect on rPLA(2). Antibody against the 42-kDa protein not only precipitated the rPLA(2) activity, but also reacted with the 42-kDa protein from bovine and human IRBCs in immunoblot analysis. The 42-kDa protein band was selectively detected in murine fetal liver cells known as a type of progenitor cells of IRBCs. It was found that EA4, a derivative of quinone newly developed as an inhibitor for rPLA2, inhibited a Ca2+ ionophore-induced AA release from human and bovine RBCs, indicating that this enzyme is responsible for the Ca-dependent AA release from mammalian RBCs. Finally, erythroid progenitor cell assay utilizing diaminobenzidine staining of hemoglobinized fetal liver cells showed that rPLA2 detectable in erythroid cells was down-regulated when differentiated to non-erythroid cells. Together, our results suggest that the 42-kDa rPLA, identified as a novel form of Ca-dependent PLA(2) may play an important role in hemostasis, thrombosis, and/or erythropoiesis through the Ca2+-dependent release of AA.