Characterization of an Ammonium Transport System in Filamentous Fungi with Methylammonium-14C As the Substrate

Abstract

Abstract Penicillium chrysogenum will grow on methylamine (methylammonium chloride) as its sole nitrogen source. The amine enters the mycelium by means of a transport system (permease) that satisfies several criteria for its identity as an ammonia (or ammonium ion) permease. Transport is temperature-dependent (Q10 of 2.1 between 20° and 30°), pH-dependent (optimum between pH 4.5 and pH 8.5), and ionic strength-dependent (optimum and relatively constant between 0 and 10-3 m KCl). Methylamine transport by nitrogen-sufficient mycelium is extremely low and obeys first order kinetics up to 10-3 m external substrate. Nitrogen starvation causes the derepression or deinhibition (or both) of a saturable transport system (Km ≃ 10-5 m, Vmax ≃ 10 µmoles per g-min). This results in an 800-fold increase in the rate of methylamine transport at external substrate concentrations below 10-5 m. Ethylamine transport has the same Vmax, but a much higher Km value (approximately 10-4 m). Methylamine transport is not inhibited significantly by other amines, amino alcohols, or amino acids previously shown to be susbtrates of a nitrogen-regulated amino acid permease (Benko, P. V., Wood, T. C., and Segel, I. H., Arch. Biochem. Biophys., 129, 498 (1968)). The amino acid permease and the methylamine permease also do not develop coincidentally during nitrogen starvation, while the choline permease is unaffected by nitrogen starvation. Ammonia (ammonium ion) is a potent competitive inhibitor of methylamine transport (Ki approximately 2.5 x 10-7 m). An independent estimate of the Vmax for ammonium transport, based on the time required to release the inhibition of methylamine-14C transport, gives a value of about 10 µmoles per g-min. Although the original development of transport activity could result from derepression, the permease is subject to feedback inhibition by intracellular glutamine and asparagine.