Molecular and General Genetics MGG

, Volume 197, Issue 3, pp 358–362 | Cite as

In vitro reconstitution of NADH: nitrate reductase in nitrate reductase-deficient mutants of barley

  • Komaratchi R. Narayanan
  • Andreas J. Müller
  • Andris Kleinhofs
  • Robert L. Warner
Article
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Summary

In vitro complementation of the nitrate reductase-deficient barley mutant nar2a extracts with molybdenum cofactor from commercial xanthine oxidase resulted in reactivation of NADH: nitrate reductase activity. Maximum reactivation was achieved with 7.5 μg/ml xanthine oxidase (final concentration), 10 mM glutathione (final concentration) and incubation for 30 min at room temperature (ca. 25°C). This in vitro complementation assay was used to determine the presence of functional apoprotein and molybdenum cofactor in 12 nitrate reductase-deficient barley mutants. Extracts of all nar1 alleles contained functional molybdenum cofactor (complemented with nar2a) but they lacked functional apoprotein (did not complement with molybdenum cofactor from xanthine oxidase). The nar2a, nar3a and nar3b extracts were able to donate functional apoprotein, but were poor sources of functional molybdenum cofactor. These data are in agreement with our previous assignment of nar1 to the barley NADH: nitrate reductase structural locus and nar2 and nar3 to molybdenum cofactor functions. Wild type cv. Steptoe barley seedlings grown in the absence of nitrate and lacking nitrate reductase activity contained low levels of molybdenum cofactor. Nitrate induction resulted in a several-fold increase in the measurable molybdenum cofactor levels that was correlated with the increase in nitrate reductase activity.

Keywords

Nitrate Glutathione NADH Nitrate Reductase Xanthine Oxidase 
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Copyright information

© Springer-Verlag 1984

Authors and Affiliations

  • Komaratchi R. Narayanan
    • 1
  • Andreas J. Müller
    • 1
  • Andris Kleinhofs
    • 1
  • Robert L. Warner
    • 1
  1. 1.Department of Agronomy and Soils and Program in Genetics and Cell BiologyWashington State UniversityPullmanUSA

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