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Proton Transporting Enzymes at the Tonoplast of Leaf Cells of the CAM Plant Kalanchoë daigremontiana. I. The ATPase

Jochem, Petra ; Lüttge, Ulrich

Journal of Plant Physiology, 1987, Vol.129(3), pp.251-268 [Periódico revisado por pares]

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  • Título:
    Proton Transporting Enzymes at the Tonoplast of Leaf Cells of the CAM Plant Kalanchoë daigremontiana. I. The ATPase
  • Autor: Jochem, Petra ; Lüttge, Ulrich
  • Assuntos: Kalanchoë Daigremontiana ; Crassulacean Acid Metabolism ; Atpase ; Proton Transport ; Tonoplast ; BSA ; Btp ; Cam ; Dccd ; Dtt ; Edta ; Hepes ; Mes ; Oxonolvi ; Pvp ; Pi ; SDS ; Tricin ; Tris
  • É parte de: Journal of Plant Physiology, 1987, Vol.129(3), pp.251-268
  • Descrição: Fractions enriched in purified tonoplast vesicles of the CAM plant Kalanchoë daigremontiana were obtained by sucrose-gradient centrifugation of mesophyll-protoplast homogenates. Nitrate-sensitive and azide-resistant ATP-hydrolysis activity at pH 8.0 was used as a tonoplast marker. Two major fractions containing this activity were obtained. First, a light one was found at 15 % sucrose in a continuous sucrose gradient (15 %/60 %) or at the 0/20 % sucrose interface of a discontinuous gradient (20 %, 33 % and 60 % sucrose from top to bottom). Secondly, a heavy one was received in the pellet of both gradients centrifuged at 100,000 g. The tonoplast vesicles showed ATP-hydrolysis activity, MgATP-dependent H + uptake as measured by quinacrine- or 9-aminoacridine-fluorescence quenching, and MgATP-dependent hyperpolarization (interior positive) as detected by Oxonol-VI-absorbance changes. ATP hydrolysis and H + transport were stimulated by chloride and malate. The experiments suggest that malate stimulation is due to both a direct effect on the tonoplast-ATPase molecule and an action of the permeant malate anion interfering with the proton electrochemical gradient across the membranes. Malate increases N0 3 - inhibition at low N0 3 - concentrations. MgATP-dependent membrane hyperpolarization was reduced in the presence of chloride and malate, providing further evidence that these ions are able to permeate the membrane. Half saturation was between 0.33 and 0.81 mM MgATP for ATP hydrolysis depending on experimental conditions and 1.90 mM for H + transport. The pH optimum was between 6.5 and 8.0 for ATP hydrolysis, depending on conditions, and 8.5 for H + transport. The role of the vacuolar membrane ATPase of K. daigremontiana for active malic-acid accumulation and its regulation is discussed.
  • Idioma: Inglês

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