Methylammonium transport in Anacystis nidulans R-2

Abstract

Methylammonium was taken up rapidly by illuminated cells of Anacystis nidulans R-2, leading to internal concentrations of 1.3 0.1 mM within 1 min, and a gradient of up to 200 between the cells and medium. Accumulation of 14CH3NH3' required at least 5 mM NaCl, but the uptake rate was independent of medium pH between 6.5 and 9. The kinetics of uptake could be resolved into an initial fast phase lasting less than 1 min (approximate Ki,, 7.2 ,uM; Vmax, 12.5 nmol minmg of protein-l at 15°C). A second, slower phase associated with product formation was eliminated by preincubation with methionine sulfoximine, a specific inhibitor of glutamine synthetase; the rapid phase was unaffected by this treatment. Ammonium ions competed with 4CH3NH3' for entry, and addition of 5 ,iM NH4' or 100 ,uM CH3NH3+ released 14CH3NH3+ accumulated during the rapid phase of entry. Small additions of NH4+ made at the same time as additions of 14CH3NH3+ delayed the start of radioactivity uptake by a time which corresponded accurately with the period needed for the complete removal of the added NH4+. The effects of inhibitors on accumulation and carbocyanine dye fluorescence suggest that ATP-dependent membrane potential was needed to drive 14CH3NH3+ transport. Spheroplasts were as active as whole cells in accumulating NH4' and 14CH3NH3', indicating that soluble

Ammonia translocation in cyanobacteria

“…With a small number of exceptions, noted below, uptake of methylammonium by both filamentous and unicellular cyanobacteria is biphasic, an initial rapid phase which lasts about 1 rain being followed by a slower, sustained uptake which may continue linearly for at least I h [4,28,29,44]. The rapid phase shows saturation kinetics, with apparent K m values usually around 10/tM (Fig.…”

“…The extent to which protein and amino acid turnover continues in growing cells is not known. Additional factors contribute to underestimation of transport rate: first, transport systems are inherently reversible, as demonstrated in the case of a cyanobacterium by exchange-diffusion experiments [28], so that it is not possible to measure accurately the rate at which individual ions move in and out of the cell using a natural substrate, initially present on both sides of the membrane. Secondly, at the intracellular pH of about 7.6 maintained by illuminated cells [6,9], approximately 1~ of the internal pool of ammonia is unprotonated and more freely permeable through the membrane than the charged ion, leading to a second mode of loss from the internal compartment, l~topic methods, which permit determination of initia~ rates, can minimize these ~3roblems, but the inconveniently short half-life of N, and the relatively large samples needed for tSN/14N ratio measurements, has restricted their use in such investigations.…”

“…The overall effect of the higher permeability will, however, be substantially offset by the smaller ratio of unprotonated to total amine in the internal environment consequent on its higher pK a (10.65 at 25°C as compared with 9.25 for NH3). A second and more controversial problem in the use of methylammonium as an analogue for ammonium in transport studies is that, although there have been no reports that methyl~mmonium can be utilized as a nitrogen source by cyanobacteria, the intracellular formation of T-methyl 81utamine by both unicellular and filamentous species has been amply demonstrated [4,27,28]. TI~s requires that a distinction be made between free and chemic~dly modified radioactive analogue associated with the cells in all experiments; this point is brought out further in ~he discussion of investigations of methylammonium uptake below.…”

“…Both equilibration and uptake measurements have commonly been carried out under illumination, and although some uptake may occur in darkness, there is general agreement that significant uptake of methylammonium at neutral, as opposed to alkaline, pH requires energy, and is driven by the membrane potential (B~) component of protonmotive force. In gynechococcus, for example, CH3NH ~" uptake was as rapid at pH 7.0 as at 7.5, where external and intracel!ular pH are equal, while transient reduction of B~/' by adding high concentrations of K +, or more permanent collapse by nigericin, is strongly inhibitory [28].…”

“…The pH of the suspending medium governs the extent to which an amine is protonated, and there-...w fore the proportion of the total molecules available for transport as the ion or through passive cL flos.aquae [44], and buffers supplemented with bicarbonate have been used for Synechoc6xus [28,45], The differences in carbon supply io cells may influence uptake kinetics, as is further discussed below. Both equilibration and uptake measurements have commonly been carried out under illumination, and although some uptake may occur in darkness, there is general agreement that significant uptake of methylammonium at neutral, as opposed to alkaline, pH requires energy, and is driven by the membrane potential (B~) component of protonmotive force.…”