title: Purification and characterization of rabbit platelet cytosolic phospholipase A2.
abstract: A phospholipase A2 was purified from rabbit platelet cytosolic fraction to near homogeneity by sequential column chromatographies on heparin-Sepharose, DEAE-Sephacel, butyl-Toyopearl, DEAE-5PW ion-exchange HPLC, and TSK gel G3000SW gel-filtration HPLC. The final preparation with an estimated specific activity of 8630 nmol/min per mg protein, showed a single band with a molecular mass of about 88 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. The 88-kDa phospholipase A2 exhibited a fatty acid preference; it hydrolyzed phospholipid bearing an arachidonoyl residue at the sn-2 position more effectively than that with a linoleoyl residue. The catalytic activity of the purified enzyme with phosphatidylcholine or phosphatidylethanolamine increased sharply in the presence of between 10(-7) and 10(-6) M calcium ion, indicating that it could be regulated by less than micromolar concentration of calcium. These characteristics differ from those of platelet secretory 14-kDa phospholipase A2 reported previously. Therefore, this 88-kDa enzyme is a novel phospholipase A2 and may participate in the stimulus-dependent release of arachidonoyl residues in rabbit platelets.
authors.affiliation: Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
authors.forename: D K
authors.initials: DK
authors.lastname: Kim
authors.forename: I
authors.initials: I
authors.lastname: Kudo
authors.forename: K
authors.initials: K
authors.lastname: Inoue
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010741:Phospholipases A / Q000097:blood* / Q000302:isolation & purification; D054467:Phospholipases A2; D011817:Rabbits
chemical_list: D003902:Detergents; D010741:Phospholipases A; D054467:Phospholipases A2