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Determination of the membrane potential of vacuoles isolated from red-beet storage tissue

Evidence for an electrogenic ATPase

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Abstract

The membrane potential of isolated vacuoles of red beet (Beta vulgaris L.) was estimated using several methods. The quenching of the fluorescence of the cyanine dyes 3,3′-diethylthiodicarbocyanine iodide (DiS-C2−(5)) and 3,3′-dipropylthiodicarbocyanine iodide (DiS-C3−(5)) in vacuoles indicated a transmembrane potential difference, negative inside at low external potassium concentrations. The Δψ was found to be-55 mV with two other methods, the distribution of 204T1+ in the presence of valinomycin and the distribution of the lipophilic cation triphenylmethylphosphonium. Uncouplers reduced this value to-35 mV. High external potassium concentrations, comparable to cytosolic values, abolished the membrane potential almost completely. The addition of 1 mM Tris-Mg2+-ATP markedly hyperpolarized the membrane to-75 mV. This effect was prevented by inhibitors of the ATPase activity located in isolated vacuole membranes.

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Abbreviations

ANS:

aminonaphthalene sulfonate

DiS-C2−(5):

3,3′-diethylthiodicarbocyanine iodide

DiS-C3−(5):

3,3′-dipropylthiodicarbocyanine iodide

EDAC:

1-ethyl-3-C-3dimethylaminopropylcarbodiimide

FCCP:

carbonylcyanide-p-trifluoromethoxyphenylhydrazone

MES:

morpholinoethylsulfonic acid

TPP+ :

tetraphenylphoshonium

TPMP:

triphenylmethylphosphonium

Tris:

tris(hydroxymethyl)aminomethane

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Correspondence to Sabine Doll.

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Doll, S., Hauer, R. Determination of the membrane potential of vacuoles isolated from red-beet storage tissue. Planta 152, 153–158 (1981). https://doi.org/10.1007/BF00391187

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Key words

  • ATPase
  • Beta
  • Membrane potential
  • Sucrose uptake
  • Tonoplast
  • Vacuole