title: Purification and properties of mercuric reductase from Yersinia enterocolitica 138A14.
abstract: A mercuric ion-reducing flavoprotein was purified from Yersinia enterocolitica 138A14 using dye matrix affinity chromatography. The purified enzyme had a characteristic absorption spectrum similar to those of flavin compounds, and FAD was detected as a part of the purified enzyme by thin-layer chromatography. Freshly purified preparations of the enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a molecular weight of 70,000. The isolated enzyme had a molecular weight of about 200,000 as determined by gel filtration and disc gel electrophoresis. These results suggest an apparently trimeric structure of the enzyme. Dithiothreitol treatment disrupted the trimer into a dimeric structure of 140,000. Along with ageing, as well as limited proteolytic digestion, the enzyme evolved to give a dimeric molecule of 105,000 composed of two identical subunits of 52,000. The combination of the purified enzyme with HgCl2, or unexpectedly with merthiolate, oxidised the NADPH, which was followed spectrophotometrically. The Km for HgCl2 was dependent on the concentration of exogenous thiol compounds. A comparison of physical properties as well as kinetic characteristics indicated that the enzyme from Y. enterocolitica 138A14 is similar to mercuric reductases isolated from other mercury-resistant bacteria.
authors: Université Louis Pasteur, Faculté de Pharmacie, Illkirch, France.
authors: M
authors: M
authors: Blaghen
authors: D J
authors: DJ
authors: Vidon
authors: M S
authors: MS
authors: el Kebbaj
mesh_terms: D002847:Chromatography, Agarose; D004591:Electrophoresis, Polyacrylamide Gel; D007120:Immunochemistry; D007526:Isoelectric Point; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013849:Thimerosal; D015008:Yersinia enterocolitica / Q000201:enzymology*
chemical_list: D008670:Metals; D013849:Thimerosal; D010088:Oxidoreductases; C021551:mercuric reductase