Effect of weak acids on amino acid transport by Penicillium chrysogenum: evidence for a proton or charge gradient as the driving force

Abstract

A variety of weak acids at and below their pK(a) are potent inhibitors of transport in Penicillium chrysogenum. The effective compounds include sorbate, benzoate, and propionate (common antifungal agents), indoleacetate (a plant hormone), acetylsalicylate (aspirin), hexachlorophene, and a yellow pigment produced by the mycelia under nutrient-deficient conditions, as well as the classical uncouplers 2,4-dinitrophenol, p-nitrophenol, and azide. The results suggest that a proton gradient or charge gradient is involved in energizing membrane transport in P. chrysogenum. The unionized form of the weak acids could discharge the gradient by diffusing through the membrane and ionizing when they reach an interior compartment of higher pH. Experiments with 2,4-dinitrophenol and p-nitrophenol established that the ionized species are not absorbed by the mycelium to any great extent. The transport inhibitors also caused a decrease in cellular adenosine 5'-triphosphate (ATP) levels, but there was no constant correlation between inhibition of transport and suppression of cellular ATP. A decrease in aeration of the mycelial suspension had the same effect on transport and ATP levels as the addition of a weak organic acid. The effects on transport rates and ATP levels were reversible. The instantaneous inhibition of [(14)C]l-leucine transport by NH(4) (-) (and vice-versa) in nitrogen-starved mycelia at pH values of 7 or below can be explained by competition for a common energy-coupling system. The inhibition is not observed in carbon-starved mycelia in which the NH(4) (+) transport system is absent or inactive (but the general amino acid transport is fully active), or in iodoacetate-treated mycelia in which the NH(4) (+) transport system has been differentially inactivated. At pH values greater than 7.0, NH(3) and HPO(4) (2-) inhibit transport, presumably by discharging the membrane proton or charge gradient. Aniline counteracts the inhibitory effect of NH(3) and HPO(4) (2-) possibly by acting as a proton reservoir or buffer within the membrane.