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Jung KM, Kim DK: Purification and characterization of a membrane-associated 48-kilodalton phospholipase A (2) in leaves of broad bean. Plant Physiol. 2000 Jul;123(3):1057-67.
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. |
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