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Journal of Biochemistry Advance Access published online on April 8, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn047
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© 2008 The Japanese Biochemical Society

Purification and Characterization of a Copper-Containing Amine Oxidase from Mycobacterium sp. Strain JC1 DSM 3803 grown on benzylamine

Hyun-Il Lee1, Young Min Kim2 and Young Tae Ro1,*

1Laboratory of Biochemistry, Graduate School of Medicine, Konkuk University, Chungju 380-701, and 2Department of Biology, Yonsei Unversity, Seoul 120-749, Korea

*Corresponding author. Prof. Young Tae Ro, Mailing address: Laboratory of Biochemistry, Graduate School of Medicine, Konkuk University, 1 Hwayang-dong, Kwangjin-gu, Seoul 143-701, Korea. Phone: 82-02-2030-7825. Fax: 82-02-2049-6192. E-mail: ytaero{at}kku.ac.kr.

Received February 12, 2008; Accepted March 26, 2008


  Abstract

A bacterial semicarbazide-sensitive amine oxidase (SSAO) was purified and characterized from Mycobacterium sp. strain JC1 DSM 3803 grown on benzylamine. During the purification procedures, the enzyme was tending to aggregate and exhibited heterogeneity in native PAGE. The heterogeneous forms having AO activity could be separated by their native molecular weights using gel-filtration chromatography. Most of the AOs behaved as dimers (Mr 150000) composed of a 75-kDa subunit, but some aggregated to form tetramers (Mr 300000). Besides their native molecular weight, subunit composition and Vmax value, both forms (dimer and tetramer) have almost identical biochemical properties (e.g. subunit size, optimum pH and temperature, activation energy, Km value on benzylamine, substrate and inhibitor specificities). When AO activity was observed by activity staining, the best oxidized substrate was benzylamine, although the AO also oxidized tyramine and histamine. The AO was strongly inhibited by semicarbazide and isoniazid, but KCN did not affect its activity. The purified enzyme was shown to contain 2.39 moles of Cu per mol of subunit, but there were no evidences of topaquinone cofactor involvement, when tested by absorption spectrum analysis and redox-cycling staining for quinoprotein detection.

Key Words: Amine oxidase, Benzylamine, Cooper-containing, Growth substrate, Semicarbazide-sensitive


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