Purification and characterization of pea seedling amine oxidase for crystallization studies.
Pea (Pisum sativum L.) seedling amine oxidase (EC 1.4.3.6) is the first amine oxidase to be crystallized that diffracts to atomic resolution (2.5 A). Extensive modifications of a published purification procedure were necessary to obtain protein that would give diffraction-quality crystals. Here we report the improved purification and also use this high-purity protein to reexamine some fundamental characteristics of pea seedling amine oxidase. The extinction coefficient at 280 nm (epsilon 1%(280)) and the molecular mass of the protein are investigated by a variety of techniques, yielding epsilon 1%(280) = 20 cm-1 and a mass 150 +/- 6 kD. In addition, the stoichiometry of the metal and organic cofactors, Cu(II) and 6-hydroxy dopa (Topa) quinone, respectively, is examined. The ratio of Cu(II):Topa:protein monomer is found to be 1:1:1.
McGuirl
M A
MA
Department of Chemistry and Biochemistry, Montana State University, Bozeman 59717.
McCahon
C D
CD
McKeown
K A
KA
Dooley
D M
DM
D006631:Amine Oxidase (Copper-Containing); D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003300:Copper / Q000032:analysis; D018360:Crystallography, X-Ray; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives / Q000032:analysis; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000201:enzymology*; D008970:Molecular Weight; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000302:isolation & purification*; D010946:Plants, Medicinal; D013053:Spectrophotometry
D004295:Dihydroxyphenylalanine; C067626:6-hydroxydopa quinone; D003300:Copper; D017886:Durapatite; D006631:Amine Oxidase (Copper-Containing); D000587:Oxidoreductases Acting on CH-NH Group Donors