| Abstrakt: |
Two membrane-bound, ascorbate-dependentb-type cytochromes were identified in etiolated bean (Phaseolus vulgarisL.) hypocotyls. Following solubilization of microsomal membranes and anion-exchange chromatography at pH 8.0, two major cytochrome peaks (P-I and P-II) were separated. Both cytochromes were reduced by ascorbate and re-oxidized by monodehydroascorbate, but P-I reduction by ascorbate was higher and saturated at far lower concentrations of ascorbate with respect to P-II. The a-band was symmetrically centered at 561 nm in P-I, but it was asymmetric in P-II with a maximum at 562 nm and shoulder at 557 nm. Ascorbate reduction of P-II, but not P-I, was inhibited by diethyl pyrocarbonate. Reduced P-II but not P-I was readily oxidized by certain ferric chelates, including FeEDTA and Fe-nitrilotriacetic acid. Purified P-I, associated with the plasma membrane, showed up as a 63-kDa glycosylated protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and behaved as a monomer of about 70 kDa during size-exclusion chromatography. P-I identified with a previously purified ascorbate-dependentb-type cytochrome of bean hypocotyl plasma membranes [P. Trost et al. (2000) Biochim Biophys Acta 1468:1-5]. Partially purified P-II, on the other hand, correlated with a heme-protein of 27 kDa in SDS-PAGE gels, was dimeric (60 kDa) during size-exclusion chromatography, and was associated with the tonoplast marker V-ATPase in sucrose gradients. The sequence of a peptide of 11 residues obtained by tryptic digestion of P-II was found to be identical to a segment of a putative cytochromeb561 ofZea maysand highly conserved in other related plant sequences, including that ofArabidopsis thalianacytochromeb561-1 (CAA18169). The biochemical features fully support the assignment of P-II cytochrome to the family ofcytochromeb561,ascorbate-dependent (CYBASC) cytochromes, which also includes cytochromeb561 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. [ABSTRACT FROM AUTHOR] |
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