Partial purification and characterization of an ascorbate-reducible b-type cytochrome from the plasma membrane of Arabidopsis thaliana leaves

A. Bérczi, R. J. Caubergs, H. Asard

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

The plant plasma membrane (PM) contains more than one b-type cytochrome. One of these proteins has a rather high redox potential (can be fully reduced by ascorbate) and is capable of transporting electrons through the PM. Four genes encoding proteins with considerable homology to the sequences of cytochrome b561 proteins in the animal chromaffin granule membrane have recently been identified in the genome of Arabidopsis thaliana. In order to characterize the cytochrome b561 located in the Arabidopsis PM, first PM vesicles were purified by aqueous polymer two-phase partitioning from the leaves of 9-week-old A. thaliana. PM proteins were solubilized by nonionic detergent, and the fully ascorbate-reducible b-type cytochrome was partially purified by anion-exchange chromatography steps. Potentiometric redox titration of the fraction, containing the fully ascorbate-reducible b-type cytochrome after the first anion-exchange chromatography step, revealed the presence of two hemes with redox potentials of 135 mV and 180 mV, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions containing the fully ascorbate-reducible b-type cytochrome after the second anion-exchange chromatography step revealed the presence of a single polypeptide band at about 120 kDa. However, heat treatment (15 min, 90°C) before electrophoresis was able to split the 120 kDa band into two bands with molecular masses of about 23 and 28 kDa. These values are lower than the apparent molecular mass for the fully ascorbate-reducible b-type cytochrome purified from Phaseolus vulgaris hypocotyls (about 52 kDa) but are in good agreement with those characteristic for the cytochrome b561 proteins purified from chromaffin granule membranes (about 28 kDa) and the four polypeptides predicted from the Arabidopsis genome (24-31 kDa).

Original languageEnglish
Pages (from-to)47-56
Number of pages10
JournalProtoplasma
Volume221
Issue number1-2
DOIs
Publication statusPublished - May 2003

Fingerprint

Cytochrome b Group
cytochromes
Arabidopsis
plasma membrane
Arabidopsis thaliana
Cell Membrane
Chromaffin Granules
Oxidation-Reduction
Anions
Chromatography
leaves
chromaffin granules
Proteins
Genome
redox potential
Hypocotyl
Peptides
Phaseolus
Membranes
polypeptides

Keywords

  • Arabidopsis thaliana
  • Ascorbate-reducible b-type cytochrome
  • Fast protein liquid chromatography
  • Plasma membrane
  • Protein purification

ASJC Scopus subject areas

  • Plant Science
  • Cell Biology

Cite this

Partial purification and characterization of an ascorbate-reducible b-type cytochrome from the plasma membrane of Arabidopsis thaliana leaves. / Bérczi, A.; Caubergs, R. J.; Asard, H.

In: Protoplasma, Vol. 221, No. 1-2, 05.2003, p. 47-56.

Research output: Contribution to journalArticle

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