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KMID : 0617320020110010263
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Journal of Pharmacetical Sceiences Ewha Womans University
2002 Volume.11 No. 1 p.263 ~ p.271
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Purification and Characterization of a Cytosolic, 42-kDa and Ca^2+ -dependent Phospholipase A_2 from Bovine Red Blood Cells
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Shin, Hae Sook
Chin, Mi-Reyoung/Kim, Jung Sun/Chung, Jin-Ho/Ryu, Chung-Kyu/Jung, Sung Yun/Kim, Dae Kyong
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Abstract
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It has become evident that a Ca^2+-dependent release ofarachidonic acid (AA) and subsequent fermation of bioac-tive lipid mediators such as prostag1andins and leukot.rienes in red blood cella (RBCa) can modify physiologicalfunctions of neighboring RBCs and platelets. Here we iden-tified a novel type of cytosolic PLA_2 in bovine and humanRBCe and purified it to apparent homogeneity with a14,000-fold purification. The purified enzyme, termedrPLA_2, hgs a molecular mass of 42 kDa and reveals bio-ehemical properties similar to group IV cPLA_2, but showadifferent profiles from cPLA_2 in several column chromatog-raphies. Moreover, rPLA_2 did not react with any of anti-cPLA_2 and anti-sPLA_2 antibodies and wae identified as anunknown protein in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis, Diva-lent metal ions tested exhibited similar effecte betweenrPLA_2 anf cPLA_2, whereas mercurials inhibited CPLA_2 buthad no effect on rPLA_2 Antibody against the 42-kDa pro-tein not only precipitated the rPLA_2 activity, but also re-acted with the 42-kDa protein from bovine and humanRBCs In Immunoblot analysis. The 42-kDa protein bandwas selectively detected in murine fetal livercells known asa type of progenitor cells of RBCs. It was found that EA4, aderivative of quinone newly developed as an inhibitor forrPLA_2, inhibited a Ca^2+ ionophore-induced AA release fromhumam and bovine RBCs, indicating that this enzyme isresponsible for the Ca^2+-dependent AA release from mam-malian RBCs. Finally, erythroid progenitor cell assay uti-lizing diaminobenzidine staining of hemoglobinized fetalliver celIs shewed that rPLA_2 detectable in erythroid cellswae down-regulated when differentiated to non-erythroidcells. Together, our results suggest that the 42-kDa rPLA_2identified as a novel form of Ca^2+-dependent PLA_2 may playan important role in hemostasis, thrombosis, and/or eryth-ropoiesis through the Ca^2+-drpendent release of AA.
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