Authors: Valeria Preger Sandra Scagliarini Paolo Pupillo Paolo Trost
Publish Date: 2004/09/09
Volume: 220, Issue: 3, Pages: 365-375
Abstract
Two membranebound ascorbatedependent btype cytochromes were identified in etiolated bean Phaseolus vulgaris L hypocotyls Following solubilization of microsomal membranes and anionexchange chromatography at pH 80 two major cytochrome peaks PI and PII were separated Both cytochromes were reduced by ascorbate and reoxidized by monodehydroascorbate but PI reduction by ascorbate was higher and saturated at far lower concentrations of ascorbate with respect to PII The αband was symmetrically centered at 561 nm in PI but it was asymmetric in PII with a maximum at 562 nm and shoulder at 557 nm Ascorbate reduction of PII but not PI was inhibited by diethyl pyrocarbonate Reduced PII but not PI was readily oxidized by certain ferric chelates including FeEDTA and Fenitrilotriacetic acid Purified PI associated with the plasma membrane showed up as a 63kDa glycosylated protein during sodium dodecyl sulfate–polyacrylamide gel electrophoresis SDS–PAGE and behaved as a monomer of about 70 kDa during sizeexclusion chromatography PI identified with a previously purified ascorbatedependent btype cytochrome of bean hypocotyl plasma membranes P Trost et al 2000 Biochim Biophys Acta 14681–5 Partially purified PII on the other hand correlated with a hemeprotein of 27 kDa in SDS–PAGE gels was dimeric 60 kDa during sizeexclusion chromatography and was associated with the tonoplast marker VATPase in sucrose gradients The sequence of a peptide of 11 residues obtained by tryptic digestion of PII was found to be identical to a segment of a putative cytochrome b561 of Zea mays and highly conserved in other related plant sequences including that of Arabidopsis thaliana cytochrome b5611 CAA18169 The biochemical features fully support the assignment of PII cytochrome to the family of cytochrome b561 ascorbatedependent CYBASC cytochromes which also includes cytochrome b561 of animal chromaffin granules The presence of a cytochrome reducing ferric chelates on the tonoplast is consistent with the role of plant vacuoles in iron homeostasis
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