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. 1984 Jun;75(2):285–289. doi: 10.1104/pp.75.2.285

Characterization of Nitrate Reductase from Corn Leaves (Zea mays cv W64A × W182E) 1

Two Molecular Forms of the Enzyme

Hiroki Nakagawa 1,2, Michel Poulle 1, Ann Oaks 1
PMCID: PMC1066898  PMID: 16663612

Abstract

The primary leaves from corn seedlings grown for 6 days were harvested, frozen with liquid N2 and extracted in a Tris buffer (pH 8.5, 250 millimolar) containing 1 millimolar dithiothreitol, 10 millimolar cysteine, 1 millimolar EDTA, 20 micromolar flavin adenine dinucleotide and 10% (v/v) glycerol. Nitrate reductase (NR) in the crude extract was stable for several days at 0°C and for several months at −80°C. The enzyme was purified using (NH4)2SO4 fractionation, brushite-hydroxyl-apatite chromatography and blue-sepharose affinity chromatography. The enzyme was eluted from the blue-sepharose column with a linear gradient of NADH (0-100 micromolar) or with 0.3 molar KNO3. About 10% of the original activity was recovered with NADH (NADH-NR). It had a specific activity of about 60 to 70 units (micromoles NO2− per minute per milligram protein). A sequential elution with NADH followed by KNO3 (0.3 molar) or KCl (0.3 molar) yielded 2 peaks. Rechromatography of each peak gave two peaks again. These results indicate that we are dealing with two forms of the same enzyme rather than two different NR proteins. The two NRs had different molecular weights as judged by chromatography on Toyopearl. The NADH-NR was more sensitive than the NO3−-NR to antibody prepared against barley leaf NR. In Ouchterlony assays a single precipitin line, with completely fused boundaries, was observed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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