Purification and characterization of amine oxidase from soybean seedlings.
A simple and rapid procedure for purification of soybean seedling amine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fractionation was purified by ion-exchange chromatography on a cellulose phosphate column and batch affinity chromatography on 6-aminohexyl-Sepharose. Cyclohexylamine, a competitive inhibitor, was utilized to elute the enzyme. A homogeneous enzyme was obtained with a yield higher than 25%, the content of minor components being < or = 2%. The M(r) estimated by gel filtration is 113,000 and 77,000 by sodium lauryl sulfate-polyacrylamide gel electrophoresis. The enzyme is a dimer and contains two Cu2+ ion per molecule. Its EPR spectrum is typical of Cu2+ in a tetragonal symmetry. The enzyme oxidizes cadaverine at high rate, the specific activity being 4.3 mukat/mg. Molecular, spectroscopic, and kinetic properties of this enzyme are reported.
Vianello
F
F
Department of Biological Chemistry, University of Padova, Italy.
Di Paolo
M L
ML
Stevanato
R
R
Gasparini
R
R
Rigo
A
A
D006631:Amine Oxidase (Copper-Containing); D000588:Amines / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D002103:Cadaverine / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011700:Putrescine / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology*; D013095:Spermidine / Q000378:metabolism; D013379:Substrate Specificity
D000588:Amines; D000596:Amino Acids; D006631:Amine Oxidase (Copper-Containing); D000587:Oxidoreductases Acting on CH-NH Group Donors; D002103:Cadaverine; D013095:Spermidine; D011700:Putrescine