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J. Biochem, 1988, Vol. 104, No. 1 131-135
© 1988 Japanese Biochemical Society

research-article

Purification and Some Properties of a Chloramphenicol Acetyltransferase Mediated by Plasmids fromVibrio anguillarum1

Shinji Masuyoshi*, Toyoji Okubo*, Matsuhisa Inoue* and Susumu Mitsuhashi**

*Laboratory of Drug Resistance in Bacteria, Gunma University School ofMedicine Maebashi, Gunma 371
**Episome Institute Fujimi-mura, Seta-gun, Gunma, 371-01

Vibrio anguillarum strains were isolated from chioramphenicol-resistant bacteria in diseasedfish. Plasmid Rms418, which confers chloramphenicol resistance, was transferred from V. anguillarum GN11379 to Escherichia coli K12 by conjugation. The Rms418- encoded chlorainphenicol acetyltransferase (CAT) [2.3.1.99] was isolated and purified to homogeneity using affinity chromatography on immobilized p-amino-chloramphenicol or ATP. The general CAT could be adsorbed by a matrix with a chioramphenicol base ligand (Zaidenzaig, Y.& Shaw, W.V. (1976) FEBS Lett. 62,266-271), but the Rms418-encoded CAT was not bound under these conditions. The specific activity of the enzyme, when measured by the spectrophotometric assay, was 71.4 units/mg protein at 37° The molecular weight of the enzyme treated with SDS and 2-mercaptoethanol wasshown to be approximately 22,000. The molecular weight of the native enzyme, as determined by gel filtration, was approximately 69,000, and the optimal pH was 7.8. The Kmvalues forchioramphenicol and CoASAc were 34.5 and 150 pM, respectively. Enzyme activity was inhibited by HgC1 p-chloromercuribenzoate (p-CMB), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and eth ylendiaminotetraacetic acid (EDTA). The half life at 53 wasapproximately 100 mm.

1 This work was supported in part by Grants-in-Aid (Nos. 211912, 311201, and 56030023) from the Ministry of Education, Science and Culture of Japan.


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