Purification and properties of a dissimilatory nitrate reductase from Haloferax denitrificans.
A membrane-bound nitrate reductase (nitrite:(acceptor) oxidoreductase, EC 1.7.99.4) from the extremely halophilic bacterium Haloferax denitrificans was solubilized by incubating membranes in buffer lacking NaCl and purified by DEAE, hydroxylapatite, and Sepharose 6B gel filtration chromatography. The purified nitrate reductase reduced chlorate and was inhibited by azide and cyanide. Preincubating the enzyme with cyanide increased the extent of inhibition which in turn was intensified when dithionite was present. Although cyanide was a noncompetitive inhibitor with respect to nitrate, nitrate protected against inhibition. The enzyme, as isolated, was composed of two subunits (Mr 116,000 and 60,000) and behaved as a dimer during gel filtration (Mr 380,000). Unlike other halobacterial enzymes, this nitrate reductase was most active, as well as stable, in the absence of salt.
Hochstein
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Planetary Biology Branch, Ames Research Center, Moffett Field, California 94035.
Lang
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D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004227:Dithionite / Q000494:pharmacology; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006216:Halobacteriaceae / Q000201:enzymology*; D006882:Hydroxyapatites; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000302:isolation & purification* / Q000378:metabolism
D006882:Hydroxyapatites; D046911:Macromolecular Substances; D004227:Dithionite; D017886:Durapatite; D009565:Nitrate Reductases; D050901:Nitrate Reductase