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Title: Isoelectric focusing and two-dimensional analysis of purified nitrate reductase from Aspergillus nidulans. Author: Steiner FX, Downey RJ. Journal: Biochim Biophys Acta; 1982 Sep 07; 706(2):203-11. PubMed ID: 6751405. Abstract: The assimilatory NADPH-nitrate oxidoreductase (EC 1.6.6.3) from Aspergillus nidulans was purified by means of affinity chromatography and analyzed by agarose isoelectric focusing and two-dimensional electrophoresis. NADPH-nitrate reductase activity was not activated by oxidation with potassium ferricyanide and was irreversibly inhibited by acrylamide. Electrophoresis of nitrate reductase in 7% polyacrylamide gels resulted in rapid loss of enzyme activity. Isoelectric focusing of purified enzyme in agarose gels resulted in the homogeneous band that exhibited NADPH-nitrate reductase, NADPH-cytochrome c reductase and reduced methyl viologen-nitrate reductase activities, which corresponded to an isoelectric point of 6.12 +/- 0.05 at 22 degrees C. Two-dimensional electrophoresis of focused nitrate reductase on SDS-polyacrylanide gel slabs yielded a single subunit of 54000 molecular weight. Acid treatment of the enzyme and subsequent isoelectric focusing resulted in a protein with a strongly acidic isoelectric point and reduced methyl viologen-nitrate reductase activity. It released another protein with a strongly basic isoelectric point which was inactive. It is postulated that the overall association of flavoprotein protomers with both heme and cytochrome b1 components confers a small net negative charge upon the native heteromultimer and accounts for its slightly acidic isoelectric point.[Abstract] [Full Text] [Related] [New Search]