Partial purification and characterization of an ascorbate-reducible b-type cytochrome from the plasma membrane of Arabidopsis thaliana leaves.
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 degrees 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 vulgarishypocotyls (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).
Bérczi
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A
Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.
Caubergs
R J
RJ
Asard
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H
D017360:Arabidopsis; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism*; D045303:Cytochromes b / Q000302:isolation & purification / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D010084:Oxidation-Reduction; D018515:Plant Leaves / Q000378:metabolism*; D012995:Solubility; D017186:Titrimetry
C001956:ascorbic acid 2-sulfate; D006418:Heme; D045303:Cytochromes b; D001205:Ascorbic Acid