ATP-dependent proton transport assay

Purified vacuolar membrane vesicles (10 μg of protein) were incubated with alexidine dihydrochloride or thonzonium bromide at the indicated concentrations for 10 min on ice. ATP-dependent proton transport was measured as follows: Fluorescence quenching of ACMA was monitored (ex 410 nm, em 490 nm) upon the addition of 0.5mM ATP and 1mM MgSO4. Initial velocities were calculated for 15 s following addition of MgATP (n=2). The apparently enhanced rate measured with benzbromarone and tolazamide was not dose-dependent and was sustained in the controls, when the drugs were added to the reaction mixture prior to the membrane vesicles.

Cell lines

In wild-type yeast cells

Preparation method

Limited solubility. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

Yeast cells expressing pHluorin were grown overnight to mid-log phase (0.4–0.6 A600/ml) and pre-incubated with 100 μM drug in 0.2% DMSO.

Applications

Treatment of yeast cells with thonzonium bromide significantly lowers the pH to (pHcyt 6.22±0.02), 10 μM thonzonium bromide reduces cell growth, with the cells developing a mild vma phenotype at 37 ℃. Moreover, thonzonium bromide completely prevents yeast growth at 50 and 100 μM concentrations.

Thonzonium Bromide is a surface active agent and an inhibitor of V-ATPase proton transport with EC50 value of 69 μM [1] [2].

Vacuolar ATPases (V-ATPases) are proton pumps that maintain pH homeostasis. V-ATPases couple the energy of ATP hydrolysis to proton transport across intracellular membranes [2].

Thonzonium Bromide is a surface active agent and an inhibitor of V-ATPase proton transport. In wild-type yeast cells, thonzonium bromide significantly decreased the cytosolic pH to 6.22. In vacuolar membrane vesicles, thonzonium bromide inhibited proton transport with EC50 value of 69 μM in a dose-dependent way. However, thonzonium bromide didn’t inhibit ATP hydrolysis, which suggested that thonzonium bromide uncoupled V-ATPase proton pumps. In wild-type yeast cells, thonzonium bromide (1 μM) didn’t inhibit cell growth. However, thonzonium bromide inhibited cell growth at 10 μM and caused a mild vma (vacuolar membrane ATPase) mutant growth phenotype. At concentrations up to 50 and 100 μM, thonzonium bromide completely inhibited yeast growth [2].

References:
[1].  Chafetz L, Greenough RC, Frank J. Thermal decomposition of thonzonium bromide. Pharm Res, 1986, 3(5): 298-301.
[2].  Chan CY, Prudom C, Raines SM, et al. Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p). J Biol Chem, 2012, 287(13): 10236-10250.