Purification and characterization of membrane-bound semicarbazide-sensitive amine oxidase (SSAO) from bovine lung.
Semicarbazide-sensitive amine oxidase (SSAO) has been purified from bovine lung microsomes in a form which is catalytically active and stable to storage. The enzyme, an integral membrane protein, was solubilized with Triton X-100 and purification was achieved, in the presence of detergent, by chromatography with Cibacron Blue 3GA-agarose, hydroxylapatite, Lens culinaris-agarose, Resource Q-FPLC and gel filtration on Superdex 200 HR-FPLC. This is the first reported procedure for the extensive purification of a membrane-bound SSAO. The purified enzyme had an apparent Mr of 400000 but exhibited microheterogeneity with SDS/PAGE and isoelectric focusing, probably as a result of its glycoprotein nature. It behaved as a tetramer with subunits with apparent Mr values of 100. Antibodies raised towards the purified enzyme cross-reacted with the enzymes from human lung and bovine plasma. Redox-cycling staining and reaction with carbonyl reagents were consistent with the presence of a quinone cofactor, possibly topa quinone. The enzyme was also shown to contain two mol of Cu/mol of enzyme and removal of half of this bound copper resulted essentially in complete inhibition of enzyme activity. In contrast to the reported behaviour of the SSAO enzymes from plasma, the bovine lung enzyme was relatively insensitive to inhibition by cyanide, copper-chelating agents and amiloride. The specificity of the bovine lung enzyme was also narrower than reported for soluble SSAO. It catalysed the oxidative deamination of benzylamine, methylamine, 2-phenylethylamine and histamine but had no significant activity towards dopamine, 5-hydroxytryptamine, tryptamine or tyramine.
Lizcano
J M
JM
Departament de Bioquimica i Biologia Molecular, Facultat de Medicina, Universitat Aut-onoma de Barcelona, Barcelona, Spain.
Tipton
K F
KF
Unzeta
M
M
D006631:Amine Oxidase (Copper-Containing) / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D010084:Oxidation-Reduction
D008565:Membrane Proteins; D006631:Amine Oxidase (Copper-Containing)