Purification and Characterization of Nitrate Reductase from the Halophile Archaebacterium Haloferax Mediterranei

Abstract

Nitrate reductase is induced in cells o f H aloferax mediterranei by the presence o f nitrate upon anaerobic conditions. This enzyme was purified more than 35-fold with a yield o f 49%. Densitograms o f polyacrylamide gel electrophoresis show the preparation to be 85% purity. The best enzyme preparation has a specific activity o f 13.6 U /m g protein. It is the first halophilic nitrate reductase that has been purified near to homogeneity. The purification consists o f five steps: An ammonium sulphate precipitation and four successive gel chromatographies with Sepharose C L-4B, calcium phosphate, DEAE-Sephacel and Sephacryl S-200. An average Mr o f 170,000 was estimated by gel chromatography and non-denaturing gel electrophoresis. Effectiveness o f electron donors, cofactors and inhibitors are reported. At low salt concentration the halophilic nitrate reductase was inactivated following first-order kinetics. The Km for nitrate depends on salt concentration and shows values in the range from 2.5 to 6.7 m M. © 1992, Walter de Gruyter. All rights reserved.