Purification and characterization of a membrane-associated 48-kilodalton phospholipase A(2) in leaves of broad bean.
Several lines of evidence indicate that phospholipase A(2) (PLA(2)) plays a crucial role in plant cellular responses through production of linolenic acid, the precursor of jasmonic acid, from membrane phospholipids. Here we report the purification and characterization of a 48-kD PLA(2) from the membrane fractions of leaves of broad bean (Vicia faba). The plant PLA(2) was purified to near homogeneity by sequential column chromatographies from the membrane extracts. The purified 48-kD protein migrated as a single band on a SDS-PAGE gel and its density correlated with the PLA(2) activity. It was further confirmed that this 48-kD protein is the PLA(2) enzyme based on immunoprecipitating the activity with a monoclonal antibody against it and purifying the enzyme to homogeneity with the antibody affinity column. The purified plant PLA(2) preferred 2-linolenoyl-sn-glycerol-3-phosphocholine (GPC) to 2-linoleoyl-GPC, 2-palmitoyl-GPC and 2-arachidonyl-GPC as substrates with a pH optimum at pH 7.0 to 8.0. The plant PLA(2) was activated by calmodulin and inhibited by pretreatment of 5,8,11, 14-eicosatetraynoic acid known as an inhibitor of mammalian PLA(2)s. The enzyme was characterized as a Ca(2+)-independent PLA(2) different from mammalian PLA(2)s. This membrane-associated and Ca(2+)-independent PLA(2) is suggested to play an important role in the release of linolenic acid, the precursor of jasmonic acid, through a signal transduction pathway.
Jung
K M
KM
Department of Environmental and Health Chemistry, College of Pharmacy, Chung-Ang University, 221 Huksuk-dong, Dongjak-ku, Seoul, 156-756 South Korea.
Kim
D K
DK
D015117:5,8,11,14-Eicosatetraynoic Acid / Q000737:chemistry; D000818:Animals; D000906:Antibodies; D002147:Calmodulin / Q000737:chemistry; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry; D007887:Fabaceae / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D018515:Plant Leaves / Q000737:chemistry; D010946:Plants, Medicinal; D011233:Precipitin Tests; D013379:Substrate Specificity
D000906:Antibodies; D002147:Calmodulin; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D015117:5,8,11,14-Eicosatetraynoic Acid; D010741:Phospholipases A