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Identification of an essential histidine residue at the active site of the tonoplast malate carrier in Catharanthus roseus cells

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Summary

The involvement of a histidyl residue in the binding or translocation step was investigated in the malate carrier at the tonoplast of Catharanthus roseus cells. The transport rate was strongly stimulated when the pH of the incubation medium was decreased from pH 7.0 to 5.0. The histidine-specific reagent diethylpyrocarbonate (DEPC) efficiently inhibited the activity of the malate carrier. Inhibition developed rapidly and was completed after 5 min at a concentration of 2 mM DEPC. The original substrate, malate, partially protected the carrier from inactivation by DEPC. Other organic acids (citrate, quinate) which are known to affect the malate transport of isolated vacuoles or tonoplast vesicles also showed protective properties. Inhibition of malate transport on tonoplast vesicles can also be achieved by photooxidation in the presence of the dye Rose Bengal. Malate also proved to protect against inactivation.

The results strongly support the notion that a histidyl residue(s) is involved either in the binding or translocation of malate and that the protonation of the histidyl residue is essential to provide a high rate of malate transport.

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This research was supported by the Centre National de la Recherche Scientifique and by a grant from the European Community (BRIDGE program). K.-J. Dietz acknowledges support by the Jubiläumsstiftung der Julius-Maximilians-Universität Würzburg, which made the stay in Toulouse possible, and the Sonderforschungsbereich 176.

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Dietz, K., Canut, H. & Marigo, G. Identification of an essential histidine residue at the active site of the tonoplast malate carrier in Catharanthus roseus cells. J. Membarin Biol. 129, 137–143 (1992). https://doi.org/10.1007/BF00219509

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Key Words

  • Catharanthus roseus
  • tonoplast, malate transport
  • histidine
  • diethylpyrocarbonate (protein modification)
  • Rose Bengal (photooxidation)