Abstract
It has become evident that a Ca(2+)-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 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(2) in several column chromatographies. Moreover, rPLA(2) did not react with any of anti-cPLA(2) and anti-sPLA(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 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 rPLA(2), inhibited a Ca(2+) ionophore-induced AA release from human and bovine RBCs, indicating that this enzyme is responsible for the Ca(2+)-dependent AA release from mammalian RBCs. Finally, erythroid progenitor cell assay utilizing diaminobenzidine staining of hemoglobinized fetal liver cells showed that rPLA(2) detectable in erythroid cells was down-regulated when differentiated to non-erythroid cells. Together, our results suggest that the 42-kDa rPLA(2) identified as a novel form of Ca(2+)-dependent PLA(2) may play an important role in hemostasis, thrombosis, and/or erythropoiesis through the Ca(2+)-dependent release of AA.
Highlights
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
After the 100,000 ϫ g supernatants and pellets were prepared from bovine RBCs, phospholipase A2 (PLA2) activity was assayed by analyzing the reaction products with thin layer chromatography using various phospholipids as described previously [21]
Despite accumulating evidence that lipid-derived bioactive mediators such as AA and its metabolites play a potential role in pathophysiology of RBCs, little is known about PLA2 as a major pathway leading to the production of the bioactive molecules from mammalian RBCs
Summary
Materials—1-Stearoyl-2-[1-14C]arachidonyl-sn-glycerol-3-phosphocholine (2-[1-14C]AA-GPC, 55.3 mCi/mmol), 1-palmitoyl-2-[1-14C]palmitoyl-sn-glycerol-3-phosphocholine (2-[1-14C]PA-GPC, 55.6 mCi/mmol), 1-palmitoyl-2-[114C]linoleoyl-sn-glycerol-3-phosphocholine (2-[1-14C]LA-GPC, 55.9 mCi/ mmol), 1-acyl-2-[1-14C]arachidonyl-sn-glycerol-3-phosphoethanolamine (2[1-14C]AA-GPE, 55.1 mCi/mmol), and [3H]arachidonic acid ([3H]AA, 204 Ci/mmol) were purchased from the radio-chemical center, Amersham Biosciences, Inc. (Buckinghamshire, UK). 1-Stearoyl-2-arachidonyl-sn-glycerol3-phosphocholine (2-AA-GPC), dithiothreitol, A23187, 3,3Ј-diaminobenzidine (DAB), methylcellulose, erythropoietin, and a Sepharose 4B-200 gel filtration column were purchased from Sigma Chemical Co. A pool of the active fractions was adjusted to 0.5 M (NH4)2SO4 and loaded onto a preparative Phenyl-5PW hydrophobic HPLC column (21.3 mm ϫ 15 cm) pre-equilibrated with buffer A containing 0.5 M (NH4)2SO4 at a flow rate of 5.0 ml/min. The active fractions from the DEAE-5PW column were pooled and injected onto a Sephacryl S-300 gel filtration column (30 mm ϫ 60 cm) pre-equilibrated with buffer A containing 0.1 M NaCl. The column was eluted with the same buffer at a flow rate of 1 ml/min. The active pool was concentrated into ϳ250 l using a Centricon 10 (Amicon Co., Beverly, MA) and injected onto a Superose 12 gel filtration FPLC column (10 mm ϫ 30 cm) pre-equilibrated with buffer A containing 0.1 M NaCl. The column was eluted with the same buffer at a flow rate of 0.5 ml/min. After 3 or 7 days, the dishes were stained for pseudoperoxidase with DAB and hydrogen peroxide as described previously [31]
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