Purification and some properties of a chloramphenicol acetyltransferase mediated by plasmids from Vibrio anguillarum.
Vibrio anguillarum strains were isolated from chloramphenicol-resistant bacteria in diseased fish. Plasmid Rms418, which confers chloramphenicol resistance, was transferred from V. anguillarum GN11379 to Escherichia coli K12 by conjugation. The Rms418-encoded chloramphenicol acetyltransferase (CAT) [EC 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 chloramphenicol 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 degrees C. The molecular weight of the enzyme treated with SDS and 2-mercaptoethanol was shown 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 Km values for chloramphenicol and CoASAc were 34.5 and 150 microM, respectively. Enzyme activity was inhibited by HgCl2, p-chloromercuribenzoate (p-CMB), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and ethylendiaminotetraacetic acid (EDTA). The half life at 53 degrees C was approximately 100 min.
Masuyoshi
S
S
Laboratory of Drug Resistance in Bacteria, Gunma University School of Medicine, Japan.
Okubo
T
T
Inoue
M
M
Mitsuhashi
S
S
D002413:Cations, Divalent; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004352:Drug Resistance, Microbial / Q000235:genetics; D004492:Edetic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D008970:Molecular Weight; D011815:R Factors; D014733:Vibrio / Q000201:enzymology / Q000235:genetics*; D020245:p-Chloromercuribenzoic Acid
D002413:Cations, Divalent; D002729:Chloromercuribenzoates; D020245:p-Chloromercuribenzoic Acid; D004228:Dithionitrobenzoic Acid; D004492:Edetic Acid; D015500:Chloramphenicol O-Acetyltransferase