Purification and Characterization of an NADH-Hexacyanoferrate(III) Reductase from Spinach Leaf Plasma Membrane

A. Bérczi, K. M. Fredlund, I. M. Moller

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Plasma membranes were purified from spinach (Spinacea oleracea L.) leaves by aqueous two-phase partitioning. The NADH-hexacyanoferrate(III) reductase was released from the membrane by Chaps solubilization and purified 360-fold by ion-exchange chromatography followed by affinity chromatography and size-exclusion chromatography on FPLC. A major band of 45 kDa and a minor contaminant of 66 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The band at 45 kDa cross-reacted with antibodies raised against an NADH-hexacyanoferrate(III) reductase from potato tuber microsomes. The native size of the enzyme was 160 kDa as determined by size-exclusion chromatography indicating that it is a tetramer. Two-dimensional gel electrophoresis, isoelectric focusing, followed by SDS-PAGE revealed three main bands of identical molecular weight with pI of 5.3-5.6. The enzyme contained about one flavin adenine dinucleotide (FAD) per 45-kDa subunit as determined by fluorescence spectroscopy, was specific for the β-hydrogen of NADH, preferred NADH over NADPH as electron donor, and preferred hexacyanoferrate(III) as electron acceptor, e.g., it reduced Fe3+-EDTA, cytochrome c, oxygen, and duroquinone at 70%, whereas FAD, flavin mononucleotide, duroquinone, and ubiquinone0 did not affect the activity.

Original languageEnglish
Pages (from-to)65-72
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume320
Issue number1
DOIs
Publication statusPublished - Jun 20 1995

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Spinacia oleracea
Cell membranes
NAD
Purification
Oxidoreductases
Electrophoresis
Cell Membrane
Flavin-Adenine Dinucleotide
Size exclusion chromatography
Sodium Dodecyl Sulfate
Gel Chromatography
Polyacrylamide Gel Electrophoresis
Electrons
Affinity chromatography
Flavin Mononucleotide
Fluorescence Spectrometry
Ion Exchange Chromatography
Electrophoresis, Gel, Two-Dimensional
Fluorescence spectroscopy
Isoelectric Focusing

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry

Cite this

Purification and Characterization of an NADH-Hexacyanoferrate(III) Reductase from Spinach Leaf Plasma Membrane. / Bérczi, A.; Fredlund, K. M.; Moller, I. M.

In: Archives of Biochemistry and Biophysics, Vol. 320, No. 1, 20.06.1995, p. 65-72.

Research output: Contribution to journalArticle

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