title: Purification and characterization of a cytosolic Ca(2+)-independent phospholipase A(2) from bovine brain.
abstract: The Ca(2+)-independent phospholipase A(2) (iPLA(2)) subfamily of enzymes is associated with arachidonic acid (AA) release and the subsequent increase in fatty acid turnover. This phenomenon occurs not only during apoptosis but also during inflammation and lymphocyte proliferation. In this study, we purified and characterized a novel type of iPLA(2) from bovine brain. iPLA(2) was purified 4,174-fold from the bovine brain by a sequential process involving DEAE-cellulose anion exchange, phenyl-5PW hydrophobic interaction, heparin-Sepharose affinity, Sephacryl S-300 gel filtration, Mono S cation exchange, Mono Q anion exchange, and Superose 12 gel filtration. A single peak of iPLA(2) activity was eluted at an apparent molecular mass of 155 kDa during the final Superose 12 gel-filtration step. The purified enzyme had an isoelectric point of 5.3 on two-dimensional gel electrophoresis (2-DE) and was inhibited by arachidonyl trifluoromethyl ketone (AACOCF(3)), Triton X-100, iron, and Ca(2+). However, it was not inhibited by bromoenol lactone (BEL), an inhibitor of iPLA(2), and adenosine triphosphate (ATP). The spot with the iPLA(2) activity did not match with any known protein sequence, as determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. Altogether, these data suggest that the purified enzyme is a novel form of cytosolic iPLA(2).
authors.affiliation: Department of Environmental and Health Chemistry, College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea.
authors.forename: Eui Man
authors.initials: EM
authors.lastname: Jeong
authors.forename: Kyong Hoon
authors.initials: KH
authors.lastname: Ahn
authors.forename: Hyung Jin
authors.initials: HJ
authors.lastname: Jeon
authors.forename: Ha Dong
authors.initials: HD
authors.lastname: Kim
authors.forename: Ho Sup
authors.initials: HS
authors.lastname: Lee
authors.forename: Sung Yun
authors.initials: SY
authors.lastname: Jung
authors.forename: Kwang Mook
authors.initials: KM
authors.lastname: Jung
authors.forename: Seok Kyun
authors.initials: SK
authors.lastname: Kim
authors.forename: Joseph V
authors.initials: JV
authors.lastname: Bonventre
authors.forename: Dae Kyong
authors.initials: DK
authors.lastname: Kim
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D054467:Phospholipases A2 / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D054467:Phospholipases A2