Purification and Characterization of a Cytosolic, 42-kDa and Ca²⁺-dependent Phospholipase A₂from Bovine 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 RBCs and platelets. Here we identified a novel type of cytosolic PLA₂ in bovine and human RBCs and purified it to apparent homogeneity with a 14,000-fold purification. The purified enzyme, termed rPLA₂, has a molecular mass of 42 kDa and reveals biochemical properties similar to group IV cPLA₂, but shows different profiles from cPLA₂ in several column chromatographies. Moreover, rPLA₂ did not react with any of anti-cPLA₂ and anti-sPLA₂ 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 rPLA₂ and cPLA₂, whereas mercurials inhibited cPLA₂ but had no effect on rPLA₂. Antibody against the 42-kDa protein not only precipitated the rPLA₂activity, but also reacted with the 42-kDa protein from bovine and human RBCs in immunoblot analysis. The 42-kDa protein band was selectively detected in murine fetal liver cells known as a type of progenitor cells of RBCs. 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 Ca²⁺-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 rPLA₂ identified as a novel form of Ca²⁺-dependent PLA₂ may play an important role in hemostasis, thrombosis, and/or erythropoiesis through the Ca²⁺-dependent release of AA.