Abstract
An improved procedure for the isolation of the cytochromeb 6/f complex from spinach chloroplasts is reported. With this preparation up to tenfold higher plastoquinol-plastocyanin oxidoreductase activities were observed. Like the complex obtained by our previous procedure, the complex prepared by the modified way consisted of five polypeptides with apparent molecular masses of 34, 33, 23, 20, and 17 kD, which we call Ia, Ib, II, III, and IV, respectively. In addition, one to three small components with molecular masses below 6 kD were now found to be present. These polypeptides can be extracted with acidic acetone. Cytochromef, cytochromeb 6, and the Rieske Fe-S protein could be purified from the isolated complex and were shown to be represented by subunits Ia + Ib, II, and III, respectively. The heterogeneity of cytochromef is not understood at present. Estimations of the stoichiometry derived from relative staining intensities with Coomassie blue and amido black gave 1:1:1:1 for the subunits Ia + Ib/II/III/IV, which is interesting in of the presence of two cytochromesb 6 per cytochromef. Cytochromef titrated as a single-electron acceptor with a pH-independent midpoint potential of +339 mV between pH 6.5 and 8.3, while cytochromeb 6 was heterogeneous. With the assumption of two components present in equal amounts, two one-electron transitions withE m(1)=−40 mV andE m(2)=−172 at pH 6.5 were derived. Both midpoint potentials were pH-dependent.
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Abbreviations
- Tris:
-
tris(hydroxymethyl)aminomethane
- SDS:
-
sodium dodecylsulfate
- SDS-PAGE:
-
SDS polyacrylamide gel electrophoresis
- MES:
-
2-(N-morpholino)ethanesulfonic acid
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Hurt, E., Hauska, G. Identification of the polypeptides in the cytochromeb 6/f complex from spinach chloroplasts with redox-center-carrying subunits. J Bioenerg Biomembr 14, 405–424 (1982). https://doi.org/10.1007/BF00743067
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DOI: https://doi.org/10.1007/BF00743067