Summary
The optical probes 9-aminoacridine, quinacrine, and bis (3-propyl-5-oxoisoxazol-4-yl) pentamethine oxonol (OX-VI) were used to measure the formation of pH and electric potential gradients in vesicles believed to be derived from corn root tonoplast membranes. Under certain conditions the quenching of fluorescence of 9-aminoacridine was shown to be quantitatively related to ΔpH, and the shift in the absorbance spectrum (measured as absorbance difference at 610 and 580 nm) of OX-VI shown to be quantitatively related to Δψ. In the absence of chloride ions, a positive interior membrane potential of approximately +100 mV was formed upon the addition of 5mm Mg/ATP to a suspension of membrane vesicles. The addition of Cl− salts reduced Δψ and stimulated the formation of ΔpH. In the presence of 50mmCl−, a ΔpH of approximately 1.1 units was established following the addition of 5mm Mg/ATP. The kinetics of Cl− activation of H+ transport could be resolved into a linear and a saturable component, with aK m for the saturable component between 4 and 5mm. Cl− inhibition of Δψ showed similar kinetics, indicating that Cl− activates electrogenic H+ transport as a permeant anion. The biphasic kinetics suggest 4 two pathways for Cl\t- permeation, with the linear component attributable to permeation through the lipid bilayer and the saturable component attributable to permeation through an anion channel. This model is supported by the effects of the anion channel blockers, 4-acetamido-4\t'-isothiocyano-2,2\t'-stilbenedisulfonic acid (SITS) and 4,4\t'-diisothiocyano-2,2\t'-stilbenedisulfonic acid (DIDS), which abolished the saturable component but not the linear component of Cl\t- stimulated H+ transport.
Cl− also stimulated ATPase activity associated with the membrane vesicles both in the absence and presence of gramicidin. In the presence of gramicidin the kinetics of Cl− stimulation of ATPase activity were adequately described by a Michaelis-menten function with aK m for Cl− between 4 and 5mm. This result indicated a direct role for Cl− in activating the H+-ATPase in addition to its apparent role as a permeant anion. The similarity inK m values for Cl− stimulation of the ATPase and for the saturable component of Cl− stimulation of H+ transport suggests that the H+-ATPase may be closely associated with a Cl− channel and that together they catalyze the nearly electroneutral transport of H+ and Cl− in vivo.
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Bennett, A.B., Spanswick, R.M. Optical measurements of ΔpH and Δψ in corn root membrne vesicles: Kinetic analysis of Cl− effects on a proton-translocating ATPase. J. Membrain Biol. 71, 95–107 (1983). https://doi.org/10.1007/BF01870678
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DOI: https://doi.org/10.1007/BF01870678