1887

Abstract

The magnitude of the proton gradient (Δμ̄) driving solute accumulation in has long been in doubt, principally because of the lack of an agreed method for assaying its electrical component, the membrane potential (Δ). In the present work, the size of the cytosine gradient (Δμ̄) that the yeast generated was used as a measure of the driving gradient (Δμ̄). The selected yeast lacked cytosine deaminase and overexpressed cytosine permease, a 1 HZcytosine system, Δμ̄, assayed in washed cell suspensions fermenting glucose and containing 0·5 or 50 mM KCI, was about 260 mV at pH 4 or 5, falling to about 194 mV at pH 7. As a first estimate, –Δμ̄ was thus at least as large at the respective pH value. A 20 mM solution of the lipophilic cation tetraphenylphosphonium lowered Δμ̄ to a value roughly equal to the magnitude of the pH gradient (ΔpH). A mathematical model was used to correct the first estimates of Δμ̄ for the effect of cytosine leakage outside the symport. In such a system, Δμ̄ cannot exceed the equivalent ratio / , where and are kinetic parameters of the symport and is the rate coefficient for leakage. The feasibility of assaying Δμ̄ depends on it not being much larger than that ratio. The model was tested successfully against observations made with yeast preparations depleted of ATP. After correction, –Δμ̄ during fermentation was estimated to be up to 25 mV larger than Δμ̄ and at least 70 mV larger than previous estimates in the literature involving lipophilic cations. From a knowledge of ΔpH, Δ was in turn deduced and compared with the maximum methylamine gradient (Δμ̄) the yeast formed. The results supported the claim in the literature that, at acid pH, Δμ̄ is a measure of Δ.

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1996-03-01
2021-05-13
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