PURIFICATION AND CHARACTERIZATION OF MEMBRANE-BOUND SEMICARBAZIDE-SENSITIVE AMINE OXIDASE (SSAO) FROM BOVINE LUNG

Semicarbazide-sensitive amine oxidase (SSAO) has been purified from bo vine lung microsomes in a form which is catalytically active and stabl e to storage. The enzyme, an integral membrane protein, was solubilize d with Triton X-100 and purification was achieved, in the presence of detergent, by chromatography with Cibacron Blue 3GA-agarose, hydroxyla patite, Lens culinaris-agarose, Resource Q-FPLC and gelfiltration on S uperdex 200 HR-FPLC. This is the first reported procedure for the exte nsive purification of a membrane-bound SSAO. The purified enzyme had a n apparent M-r of 400 000 but exhibited microheterogeneity with SDS/PA GE and isoelectric focusing, probably as a result of its glycoprotein nature. It behaved as a tetramer with subunits with apparent M-r value s of 100. Antibodies raised towards the purified enzyme cross-reacted with the enzymes from human lung and bovine plasma. Redox-cycling stai ning and reaction with carbonyl reagents were consistent with the pres ence 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 t his bound copper resulted essentially in complete inhibition of enzyme activity, In contrast to the reported behaviour of the SSAO enzymes f rom plasma, the bovine lung enzyme was relatively insensitive to inhib ition by cyanide, copper-chelating agents and amiloride. The specifici ty of the bovine lung enzyme was also narrower than reported for solub le SSAO. It catalysed the oxidative deamination of benzylamine, methyl amine, 2-phenylethylamine and histamine but had no significant activit y towards dopamine, 5-hydroxytryptamine, tryptamine or tyramine.