Purification and characterization of amine oxidase from pea seedlings.
A novel, simple, and rapid procedure for the purification of pea seedling amine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fractionation, was purified in two steps: the first one by anion-exchange chromatography and the second one by affinity chromatography. The first chromatography step was carried out on a diethylaminoethyl-cellulose column. By lowering the amount of protein loaded on the column and the buffer concentration it was possible to obtain an enzyme pure at 95% (sp act 1.2 microkat/mg). To achieve a higher degree of purification various affinity resins were prepared and tested. The resins were obtained by covalent immobilization of polyamines on Sepharose according to three different procedures. The best results were obtained with 6-aminohexyl-Sepharose 2B, prepared using CNBr as coupling agent, and eluting the enzyme by a solution containing 1, 4-diaminocyclohexane. This last compound was found to be a relatively strong competitive inhibitor of the oxidative deamination of cadaverine catalyzed by pea seedling amine oxidase (Ki = 32 microM). According to this procedure an electrophoretically homogeneous enzyme, characterized by a specific activity of 1.63 microkat/mg, was obtained.
Vianello
F
F
Department of Biological Chemistry, University of Padua, Padova, Italy.
Malek-Mirzayans
A
A
Di Paolo
M L
ML
Stevanato
R
R
Rigo
A
A
D006631:Amine Oxidase (Copper-Containing) / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000588:Amines / Q000494:pharmacology; D000645:Ammonium Sulfate; D002103:Cadaverine / Q000378:metabolism; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003514:Cyclohexylamines / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018532:Pisum sativum / Q000201:enzymology*; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D012685:Sepharose; D013053:Spectrophotometry
D000588:Amines; D003514:Cyclohexylamines; D004791:Enzyme Inhibitors; D010940:Plant Proteins; C088969:1,4-diaminocyclohexane; D012685:Sepharose; D006631:Amine Oxidase (Copper-Containing); D002103:Cadaverine; D000645:Ammonium Sulfate