Partial purification and characterization of an ascorbate-reducible b-type cytochrome from the plasma membrane of Arabidopsis thaliana leaves |
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Authors: | Bérczi A Caubergs R J Asard H |
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Institution: | (1) Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged, HU;(2) Department of Biology, University of Antwerp, Antwerp, BE;(3) Beadle Center for Genetic Research, University of Nebraska-Lincoln, Lincoln, Nebraska, US |
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Abstract: | Summary. 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
b
561 proteins in the animal chromaffin granule membrane have recently been identified in the genome of Arabidopsis thaliana. In order to characterize the cytochrome b
561 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 b
561 proteins purified from chromaffin granule membranes (about 28 kDa) and the four polypeptides predicted from the Arabidopsis
genome (24–31 kDa).
Received May 4, 2002; accepted July 26, 2002; published online May 21, 2003
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ID="*" Correspondence and reprints: Institute of Biophysics, BRC, Hungarian Academy of Sciences, POB 521, 6701 Szeged, Hungary. |
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Keywords: | : Arabidopsis thaliana Ascorbate-reducible b-type cytochrome Fast protein liquid chromatography Plasma membrane Protein purification |
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