The assay of the molybdenum cofactor in higher plants as affected by pyridine nucleotides and nitrate

N. A. Savidov, M. Sagi, S. H. Lips

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

The molybdenum cofactor (MoCo) is the prosthetic group of several enzymes in higher plants including nitrate reductase (NR, EC 1.6.6.1). The traditional assay of MoCo includes its release from binding proteins, followed by its complementation with the NR (EC 1.6.6.3) apoprotein of the nit-I mutant of Neurospora crassa and the assay of the assembled NR. NADPH added daring complementation allowed reduction of nitrate, present in the crude extract of barley and ryegrass, to nitrite by the reconstituted NR. Thus, the extent of nitrite measured during the assay was a result not only of MoCo content but also of the nitrate pool of the plant tissue. This error resulted in overestimations of MoCo content in nitrate-fed plants of barley and ryegrass. Exclusion of NADPH from the complementation medium eliminated nitrite accumulation during this stage of the assay. NADPH was unnecessary as a stabilizing factor of MoCo and the hybrid NR during complementation. NADH and nitrate in the complementation medium enhanced reconstituted NR activity while oxidized pyridine nucleotides applied at relatively high concentrations strongly inhibited NR activity. Nitrate concentrations in the complementation medium should be standardized to avoid erroneous estimations of MoCo.

Original languageEnglish
Pages (from-to)419-426
Number of pages8
JournalPlant Physiology and Biochemistry
Volume35
Issue number6
StatePublished - 1 Jan 1997

Keywords

  • Hordeum vulgate
  • Lolium multiflorum
  • Molybdenum cofactor
  • Nitrate
  • Nitrate reductase

ASJC Scopus subject areas

  • Physiology
  • Genetics
  • Plant Science

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