Plant Physiol, July 2000, Vol. 123, pp. 1057-1068

Purification and Characterization of a Membrane-Associated 48-Kilodalton Phospholipase A₂ in Leaves of Broad Bean¹

Department of Environmental and Health Chemistry, College of Pharmacy, Chung-Ang University, 221 Huksuk-dong, Dongjak-ku, Seoul, 156-756 South Korea

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.