Purification and characterization of a membrane-associated 48-kilodalton phospholipase A2 in leaves of broad bean

Abstract

Several lines of evidence indicate that phospholipase A2 (PLA2) 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 PLA2 from the membrane fractions of leaves of broad bean (Vicia faba). The plant PLA2 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 PLA2 activity. It was further confirmed that this 48-kD protein is the PLA2 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 PLA2 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 PLA2 was activated by calmodulin and inhibited by pretreatment of 5,8,11,14-eicosatetraynoic acid known as an inhibitor of mammalian PLA2s. The enzyme was characterized as a Ca2+-independent PLA2 different from mammalian PLA2s. This membrane-associated and Ca2+-independent PLA2 is suggested to play an important role in the release of linolenic acid, the precursor of jasmonic acid, through a signal transduction pathway.