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Characteristics, partial purification and reconstitution of the vacuolar malate transporter of the CAM plant Kalanchoe daigremontiana Hamet et Perrier de la Bathie

Ratajczak , R. ; Kemna , I. ; Luttge , U.

Planta, 1994, Vol.195(2), pp.226-236 [Periódico revisado por pares]

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  • Título:
    Characteristics, partial purification and reconstitution of the vacuolar malate transporter of the CAM plant Kalanchoe daigremontiana Hamet et Perrier de la Bathie
  • Autor: Ratajczak , R. ; Kemna , I. ; Luttge , U.
  • Assuntos: Tonoplast ; Nutrient Transport ; Chemical Constituents Of Plants ; Vacuoles ; Bryophyllum Daigremontianum ; Purification ; Crassulacean Acid Metabolism ; Protein Content ; Malic Acid
  • É parte de: Planta, 1994, Vol.195(2), pp.226-236
  • Notas: Includes references
  • Descrição: When native tonoplast vesicles of Kalanchoe daigremontiana Hamet et Perrier de la Bathie were energized by an artificial K+ gradient establishing only an inside-positive electrical membrane potential (delta psi), it was shown that delta psi was sufficient as the sole driving force and that a proton gradient (delta pH) is not required for malate uptake. Following [14C]malate uptake, Km-malate of the malate transporter was estimated as 2.7-3.0 mM, a value that would allow malate synthesis via phosphoenolpyruvate carboxylase and malate accumulation in vivo in view of feed-back inhibition of cytosolic phosphoenolpyruvate carboxylase by malate. The maximum reaction velocity (Vmax) was found to be between 30 and 85 nmol malate.min-1.mg protein-1, a value that would explain nocturnal malate accumulation in K. daigremontiana even if the transporter were operating below substrate saturation. Citrate (50 mM at pH 7) inhibited transport by 78%. The malate-transport protein of the tonoplast of K. daigremontiana may be a carboxylate uniporter with strong affinities for malate and citrate. From total tonoplast proteins solubilized from native tonoplast vesicles the malate transporter was functionally reconstituted into phospholipid liposomes. The malate transporter was purified and separated from the tonoplast H+-ATPase by hydroxyapatite chromatography, but not from the tonoplast H+-pyrophosphatase. The partially purified malate-transport protein was functionally reconstituted into phospholipid liposomes. In these final proteoliposomes, 0.6% of the protein of the initial tonoplast-vesicle preparation used for solubilization of membrane proteins was recovered. Using the specific rates of malate transport as a reference, i.e. rates of transport related to protein in the preparations, enrichment of the malate transporter in the final proteoliposomes obtained with the reconstitution of the hydroxyapatite eluate was 44-fold compared to the initial native tonoplast vesicles and 2000-fold compared to the liposomes reconstituted from solubilized tonoplast proteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the peptides from the final proteoliposomes, which were functional in malate transport, showed only a few polypeptide bands among which the malate transporter must be found. ; Includes references ; p. 226-236.
  • Idioma: Inglês

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