University of Illinois Urbana-Champaign

Evolution of Nitrogen Oxide(s) From Soybean and the Association of This Activity to the Constitutive Nad(p)h-Nitrate Reductase Enzyme

Dean, John Vahle

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Description

Title
Evolution of Nitrogen Oxide(s) From Soybean and the Association of This Activity to the Constitutive Nad(p)h-Nitrate Reductase Enzyme
Author(s)
Dean, John Vahle
Issue Date
1988
Doctoral Committee Chair(s)
Harper, James E.
Department of Study
Agronomy
Discipline
Agronomy
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
  • Agriculture, Agronomy
  • Biology, Plant Physiology
Abstract
  • Gas chromatography and mass spectrometry were used to determine the identity and the quantity of volatile N products produced during the helium-purged in vivo nitrate reductase (NR) assay of soybean (Glycine max (L.) Merr. cv Williams) and winged bean (Psophocarpus tetragonolobus (L.) DC. cv Lunita) leaflets. Nitric oxide (NO) and nitrous oxide (N$\sb2$O) were identified as the main N products formed. NO and N$\sb2$O produced from soybean leaflets were both labeled with $\sp $N when $\sp $N-nitrate was used in the assay medium, demonstrating that both were produced from nitrate during nitrate reduction. Leaves of winged bean, subjected to the purged in vivo NR assay, evolved greater quantities of NO and N$\sb2$O than did soybean. In both species NO production was dominant. In contrast, with similar assays, NO and N$\sb2$O were not evolved from leaves of the nr$\sb1$ soybean mutant which lacks the constitutive NR enzymes.
  • A two step purification protocol was used in an attempt to separate the constitutive NAD(P)H-nitrate reductase activity from the NO$\sb{\rm (x)}$ (nitric oxide and nitrogen dioxide) evolution activity extracted from soybean leaflets. Both of these activities co-elute with NADPH from Blue Sepharose columns. None of the fractions eluted with NADH were found to contain NO$\sb{\rm (X)}$ activity. FPLC-anion exchange chromatography following Blue Sepharose affinity chromatography was also unable to separate the two activities. These data provide strong evidence that the constitutive NAD(P)H-NR (pH 6.5) in soybean is the enzyme responsible for NO$\sb{\rm (X)}$ formation.
  • The Blue Sepharose purified soybean enzyme has a pH optimum of 6.75, an apparent K$\sb{\rm m}$ for nitrate of 0.49 mM, and an apparent K$\sb{\rm m}$ for NADPH and NADH of 7.2 and 7.4 $\mu$M, respectively, for the NO$\sb{\rm (x)}$ activity. In addition to NAD(P)H, the enzyme can also use FMNH$\sb2$ and reduced methyl viologen (MV) as electron donors for NO$\sb{\rm (x)}$ activity. The NADPH-, FMNH$\sb2$-, and MV-NO$\sb{\rm (x)}$ activities were all inhibited by cyanide. The NADPH activity was also inhibited by p-hydroxymercuribenzoate, whereas, the FMNH$\sb2$ and MV activities were relatively insensitive to inhibition. These data indicate that the terminal molybdenum-containing portion of the enzyme is involved in the reduction of nitrate to NO$\sb{\rm (x)}$.
Graduation Semester
1988
Type of Resource
text
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http://hdl.handle.net/2142/71647

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