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

• Published in: Zeitschrift für Naturforschung C. A journal of biosciences Vol. 47; no. 9; pp. 670 - 676
• Main Authors: Alvarez-Ossorio, María C., Muriana, Francisco J. G., Rosa, Francisco F. de la, Relimpio, Angel M.
• Format: Journal Article
• Language: English
• Published: Tübingen Verlag der Zeitschrift für Naturforschung 01-10-1992
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Enzyme Characterization; Enzyme Purification; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Haloferax mediterranei; Halophilic Nitrate Reductase; Oxidoreductases; Purification; Archaeobacteria; Enzyme; Nitrate reductase; Haloferax mediterranei; Bacteria; Halobacteriaceae; Physicochemical properties
• ISSN: 0939-5075; 1865-7125
• DOI: 10.1515/znc-1992-9-1005

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