Purification of nitrate reductase from Nicotiana plumbaginifolia by affinity chromatography using 5'AMP-Sepharose and monoclonal antibodies

• Published in: European Journal of Biochemistry Vol. 179; no. 3; pp. 617 - 620
• Main Authors: MOUREAUX, T, LEYDECKER, M.-T, MEYER, C
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell 15-02-1989; Wiley
• Subjects: Analytical, structural and metabolic biochemistry; Antibodies, Monoclonal; Biochemistry, Molecular Biology; Biological and medical sciences; Blotting, Western; Chromatography, Affinity - methods; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; In Vitro Techniques; Life Sciences; Nicotiana - enzymology; Nicotiana plumbaginifolia; Nitrate Reductase; Nitrate Reductases - genetics; Nitrate Reductases - isolation & purification; Oxidoreductases; Plants, Toxic; Sepharose; Nitrate reductase (NADH); Purification; Immunoaffinity chromatography; Enzyme; Monoclonal antibody; Nicotiana plumbaginifolia; Ribulosebisphosphate carboxylase; Affinity chromatography; Dicotyledones; Angiospermae; Plant origin; Spermatophyta; Solanaceae; BIOLOGIE MOLECULAIRE
• ISSN: 0014-2956; 1432-1033; 1432-1327
• DOI: 10.1111/j.1432-1033.1989.tb14591.x

Nitrate reductase was purified from leaves of Nicotiana plumbaginifolia using either 5'AMP-Sepharose chromatography or two steps of immunoaffinity chromatography involving monoclonal antibodies directed against nitrate reductase from maize and against ribulose-1,5-bisphosphate carboxylase from N. plumbaginifolia. Nitrate reductase obtained by the first method was purified 1000-fold to a specific activity of 9 units/mg protein. The second method produced an homogenous enzyme, purified 21,000-fold to a specific activity of 80 units/mg protein. SDS/PAGE of nitrate reductase always resulted in two bands of 107 and 99.5 kDa. The 107-kDa band was the nitrate reductase subunit of N. plumbaginifolia; the smaller one of 99.5 kDa is thought, as commonly reported, to result from proteolysis of the larger protein. The molecular mass of 107 kDa is close to the values calculated from the coding sequences of the two nitrate reductase genes recently cloned from tobacco (Nicotiana tabacum cv Xanthi).