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

摘要: When native tonoplast vesicles of Kalanchoe daigremontiana Hamet et Perrier de la Bâthie were energized by an artificial K+ gradient establishing only inside-positive electrical membrane potential (ΔΨ), it was shown that ΔΨ sufficient as the sole driving force and a proton (ΔpH) is not required for malate uptake. Following [14C]malate uptake, Km-malate transporter estimated 2.7–3.0 mM, value would allow synthesis via phosphoenolpyruvate carboxylase accumulation in vivo view feed-back inhibition cytosolic malate. The maximum reaction velocity (Vmax) found to be between 30 85 nmol malate·min−1·mg protein −1 , explain nocturnal K. even if operating below substrate saturation. Citrate (50 mM at pH 7) inhibited transport 78%. malate-transport may carboxylate uniporter with strong affinities citrate. From total proteins solubilized from functionally reconstituted into phospholipid liposomes. purified separated H+-ATPase hydroxyapatite chromatography, but H+-pyrophosphatase. partially In these final proteoliposomes, 0.6% initial tonoplast-vesicle preparation used solubilization recovered. Using specific rates reference, i.e. related preparations, enrichment proteoliposomes obtained reconstitution eluate 44-fold compared 2000-fold liposomes proteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis peptides which functional transport, showed few polypeptide bands among must found.