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Isoelectric focusing and two-dimensional analysis of purified nitrate reductase from Aspergillus nidulans.
Biochim Biophys Acta. 1982 Sep 07; 706(2):203-11.BB

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.

Authors

No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

6751405

Citation

Steiner, F X., and R J. Downey. "Isoelectric Focusing and Two-dimensional Analysis of Purified Nitrate Reductase From Aspergillus Nidulans." Biochimica Et Biophysica Acta, vol. 706, no. 2, 1982, pp. 203-11.
Steiner FX, Downey RJ. Isoelectric focusing and two-dimensional analysis of purified nitrate reductase from Aspergillus nidulans. Biochim Biophys Acta. 1982;706(2):203-11.
Steiner, F. X., & Downey, R. J. (1982). Isoelectric focusing and two-dimensional analysis of purified nitrate reductase from Aspergillus nidulans. Biochimica Et Biophysica Acta, 706(2), 203-11.
Steiner FX, Downey RJ. Isoelectric Focusing and Two-dimensional Analysis of Purified Nitrate Reductase From Aspergillus Nidulans. Biochim Biophys Acta. 1982 Sep 7;706(2):203-11. PubMed PMID: 6751405.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Isoelectric focusing and two-dimensional analysis of purified nitrate reductase from Aspergillus nidulans. AU - Steiner,F X, AU - Downey,R J, PY - 1982/9/7/pubmed PY - 1982/9/7/medline PY - 1982/9/7/entrez SP - 203 EP - 11 JF - Biochimica et biophysica acta JO - Biochim Biophys Acta VL - 706 IS - 2 N2 - 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. SN - 0006-3002 UR - https://www.unboundmedicine.com/medline/citation/6751405/Isoelectric_focusing_and_two_dimensional_analysis_of_purified_nitrate_reductase_from_Aspergillus_nidulans_ L2 - https://linkinghub.elsevier.com/retrieve/pii/0167-4838(82)90488-5 DB - PRIME DP - Unbound Medicine ER -