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.