title: A wounding-induced PPO from cowpea (Vigna unguiculata) seedlings.
abstract: Polyphenol oxidases (PPO) are induced in cowpea plants by wounding. The highest activity levels were detected 48h after this stimulus in both wounded and neighbor-to-wounded unifoliates of cowpea seedlings; the increase of activity was in the order of 13 to 15-fold, respectively, in comparison to control unifoliates. Multiple molecular forms of active PPO (Mrs 58, 73 and congruent with220kDa) were detected by partially denaturing SDS-PAGE. Wounding-induced cowpea PPO were extracted and purified through (NH4)2SO4 precipitation and ion-exchange chromatography. The effects of substrate specificity, pH, thermal stability and sensitivity to various inhibitors - resorcinol, EDTA, sodium azide and tropolone - of partially purified soluble PPO were investigated. Purified wounding-induced cowpea PPO (wicPPO) showed the highest activities towards 4-methylcatechol (Km=9.86mM, Vmax=24.66 EU [DeltaAmin(-1)]) and catechol (Km=3.44mM, Vmax=6.64 EU [DeltaAmin(-1)]); no activity was observed towards l-tyrosine, under the assay conditions used. The optimum pH for wound-induced cowpea PPO was 6.0 with 4-methylcatechol as substrate. The enzyme was optimally activated by 10 mM SDS and was highly stable even after 5 min at 80 degrees C. The most effective inhibitor was tropolone, whereas addition of 10mM of resorcinol, EDTA and sodium azide were able to reduce PPO activities by 40%, 15% and 100%, respectively.
authors.affiliation: Laboratório de Química e Função de Proteínas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil.
authors.forename: Márcio S T
authors.initials: MS
authors.lastname: Pinto
authors.forename: Fabrício P
authors.initials: FP
authors.lastname: Siqueira
authors.forename: Antônia E A
authors.initials: AE
authors.lastname: Oliveira
authors.forename: Kátia V S
authors.initials: KV
authors.lastname: Fernandes
mesh_terms: D004156:Catechol Oxidase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007887:Fabaceae / Q000201:enzymology*; D007700:Kinetics; D010935:Plant Diseases; D010936:Plant Extracts / Q000378:metabolism; D036226:Seedlings / Q000201:enzymology*
chemical_list: D010936:Plant Extracts; D004156:Catechol Oxidase