Quaternary structure and composition of squash NADH:nitrate reductase

• Published in: The Journal of biological chemistry Vol. 260; no. 6; pp. 3380 - 3385
• Main Authors: Redinbaugh, M G, Campbell, W H
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25-03-1985; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acids - analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; chemicophysical properties; composicion; composition; cucurbita maxima; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Macromolecular Substances; Molecular Weight; Nitrate Reductase (NADH); Nitrate Reductases - analysis; Oxidoreductases; oxidorreductasas; oxydoreductase; Plants - enzymology; propiedades fisico quimicas; propriete physico chimique; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Nitrate reductase (NADH); Vegetals; Molecular structure; Affinity chromatography; Enzyme; Dicotyledones; Angiospermae; Cucurbitaceae; Quaternary structure; Gel filtration; Cucurbita maxima; Spermatophyta
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)83632-3

nitrate reductase (EC 1.6.6.1) was isolated from squash cotyledons (Cucurbita maxima L.) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on Bio-Gel A-1.5m. These preparations gave a single protein staining band (Mr = 115,000) on sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is homogeneous. The native Mr of nitrate reductase was found to be 230,000, with a minor form of Mr = 420,000 also occurring. These results indicate that the native nitrate reductase is a homodimer of Mr = 115,000 subunits. Acidic amino acids predominate over basic amino acids, as shown both by the amino acid composition of the enzyme and an isoelectric point for nitrate reductase of 5.7. The homogeneous nitrate reductase had a UV/visible spectrum typical of a b-type cytochrome. The enzyme was found to contain one each of flavin (as FAD), heme iron, molybdenum, and Mo-pterin/Mr = 115,000 subunit. A model is proposed for squash nitrate reductase in which two Mr = 115,000 subunits are joined to made the native enzyme. Each subunit contains 1 eq of FAD, cytochrome b, and molybdenum/Mo-pterin.