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NADH-dependent Fe3+EDTA and oxygen reduction by plasma membrane vesicles from barley roots

Authors

  • Wolfgang Brüggemann,

    Corresponding author
    1. Dept of Anatomy and Cell Biology, Philipps Univ., Robert-Koch-Str. 6, D-3550 Marburg, FRG
      W. Brüggemann (corresponding author)
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    • 1

      Dept Plant Physiology, Biological Centre, Univ. of Groningen, FOB 14, 9750 A A Haren (Gn.), The Netherlands

  • Petra R. Moog

    1. P. R. Moog, Faculty of Biology, Univ. of Oldenburg, FOB 2503, D2900 Oldenburg, FRG.
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W. Brüggemann (corresponding author)

Abstract

Brüggemann, W. and Moog, P. R. 1989. NADH-dependent Fe3+EDTA and oxygen reduction by plasma membrane vesicles from barley roots.

Biochemical properties of pyridine-dinucleotide-dependent Fe3+-EDTA reductase were analysed in purified plasma membranes (PM) from barley (Hordeum vulgare L. cv. Marinka) roots. The enzymatic activity preferred NADH over NADPH as electron donor and it was 3-fold increased in the presence of detergent. The reductase showed a pH optimum of 6.8 and saturable kinetics for NADH with Km (NADH) of 125 μM and Vmax of 143 nmol Fe (mg protein)-1 min-1 in the presence of 500 μM Fe3+EDTA. For the dependence of the reaction rate on the iron compound, Km(Fe3+EDTA) of 120 μM and Vmax of 184 nmol (mg protein)-1 min-1 were obtained. The activity was insensitive to superoxide dismutase (SOD; EC 1.15.1.1), catalase (EC 1.11.1.6) and antimycin A, but stimulated by an oxygen-free reaction medium. It could be solubilized by 0.25% (w/v) Triton X-100. The solubilized enzyme revealed one band in native polyacrylamide gel electrophoresis (PAGE) and in isoelectric focussing (IEF) at pl 7.4 by enzyme staining. Major polypeptides with molecular weights of 94, 106, 120 and 205 kDa corresponded to the enzyme-stained band from native PAGE.

Analysis of oxygen consumption by the membranes revealed the existence of NADH:CK oxidoreductase activity, which was stimulated by salicylhydroxamic acid (SHAM), chinhydron, Fe3+EDTA and Fe3+EDTA but not by K3 [Fe(CN)6] or K4[Fe (CN)6). The stimulating effect of the iron chelates on oxygen consumption was due to Fe2+ and could be suppressed by bathophenanthroline disulfonate (BPDS), SOD and p-chloromercurophenylsulfonic acid (PCMS). The results are discussed with respect to the nature of the stimulation.

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