Characterization and solubilization of residual redox activity in salt-washed and detergent-treated plasma membrane vesicles from spinach leaves

A. Bérczi, I. M. Møller

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4 Citations (Scopus)

Abstract

An NADH-hexacyanoferrate(III) oxidoreductase (N-HCF-OR) was purified from spinach leaf plasma membrane (PM) vesicles; detailed biochemical analyses, however, revealed that the purified protein is an NADH-monodehydroascorbate oxidoreductase (N-MDA-OR) located on the cytoplasmic surface of the PM. After removing all N-MDA-OR activity from the PM vesicles by consecutive treatments with hypoosmotic shock, salt, and detergents, the remaining PM (the 'stripped' PM, SPM) fraction contained about 50% of the protein and 15% of the N-HCF-OR activity of the original PM fraction. The highest redox activity (100%) of the SPM fraction was obtained with NADH as electron donor and hexacyanoferrate(III) (HCF) as electron acceptor, although redox activity could be measured also with ubiquinone-0 (23%), dichlorophenolindophenol (16%), cytochrome c (9%), and Fe3+-EDTA (2%) as electron acceptors. The following K(m) values were obtained for the N-HCF-OR activity of SPM: K(m)(NADH) = 66.5 ± 3.8 μM [with 200 μM HCF(III)], K(m)[HCF(III)] = 11.1 ± 1.1 μM (with 150 μM NADH). NAD+ competitively inhibited the activity. Under special conditions, SB-16 (palmityl sulfobetaine, a zwitterionic detergent with a C-16 hydrocarbon chain) solubilized about 50% of the protein and more than 90% of the N-HCF-OR activity of the SPM fraction. Redox activity of the solubilized fraction with dichlorophenolindophenol as electron acceptor was 45% of that with HCF(III). The SB-16-solubilized fraction contained b-type cytochrome(s) which could be reduced by dithionite > ascorbate >> NADH. Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the SB-16-solubilized SPM fraction revealed numerous polypeptides between 17 and 95 kDa. Further purification steps are needed to match the redox activities and spectrophotometric data to one or more of the polypeptides seen on the gel.

Original languageEnglish
Pages (from-to)59-65
Number of pages7
JournalProtoplasma
Volume205
Issue number1-4
Publication statusPublished - Dec 1 1998

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Keywords

  • Detergent solubilization
  • Plasma membrane
  • Redox activity

ASJC Scopus subject areas

  • Plant Science
  • Cell Biology

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