title: Biochemical characterisation of prolyl aminopeptidase from shoots of triticale seedlings and its activity changes in response to suboptimal growth conditions.
abstract: Prolyl aminopeptidase (PAP) was isolated from the shoots of three-day-old triticale seedlings and was purified using a five-step purification procedure (acid precipitation, gel filtration, anion-exchange chromatography, hydrophobic chromatography and rechromatography). The enzyme was purified 460-fold with a recovery of 6%. Prolyl aminopeptidase appears to be a tetramer consisting of four subunits, each with a molecular weight of approximately 54kDa. Its pH and temperature optimum are pH 7.5 and 37°C, respectively. The enzyme prefers substrates with Pro and Hyp at the N-terminus, but is also capable of hydrolysing β-naphthylamides (β-NA) of Ala, Phe, and Leu. The K(m) value of PAP against Pro-β-NA was the lowest among the substrates tested and it was 1.47×10(-5)M. The activity of PAP was not inhibited by EDTA, 1,10-phenantroline, or pepstatin A. The most effective inhibitors were DFP, Pefabloc, and PMSF, which are serine protease inhibitors. However, significant inhibition was also observed in the presence of E-64, which modifies sulfhydryl groups. A significant increase of the aminopeptidase activity against Pro-β-NA was observed in shoots of triticale plants grown under salinity, drought stress, and in the presence of cadmium and aluminium ions in the nutrient solution.
authors.affiliation: Department of Biochemistry, Warsaw University of Life Sciences - SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland.
authors.forename: Urszula
authors.initials: U
authors.lastname: Szawłowska
authors.forename: Edyta
authors.initials: E
authors.lastname: Zdunek-Zastocka
authors.forename: Wiesław
authors.initials: W
authors.lastname: Bielawski
mesh_terms: D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D055864:Droughts; D002523:Edible Grain / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development / Q000502:physiology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D019216:Metals, Heavy / Q000494:pharmacology; D008970:Molecular Weight; D009281:Naphthalenes / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D018520:Plant Shoots / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development / Q000502:physiology; D011480:Protease Inhibitors / Q000494:pharmacology; D021122:Protein Subunits / Q000378:metabolism; D054712:Salinity; D036226:Seedlings / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development / Q000502:physiology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D019216:Metals, Heavy; D009281:Naphthalenes; D010940:Plant Proteins; D011480:Protease Inhibitors; D021122:Protein Subunits; C072587:2-naphthylamide; D000626:Aminopeptidases; C022450:prolyl aminopeptidase