Characterization of ammonium (methylammonium)/potassium antiport in Escherichia coli.
The energetics of ammonium ion transport by Escherichia coli have been studied using [14C]methylammonium as a substrate. Rapid assays for uptake allowed kinetic parameters (CH3NH3+ Km = 36 microM; Vmax = 4 nmol X s-1 X mg-1 to be determined in the absence of CH3NH3+ metabolism. Cells cultured in media containing 1 mM NH4+ failed to express CH3NH3+ transport activity. Methylammonium accumulated at levels which were 100-fold higher than those of the medium. This accumulation was dependent upon the addition of glucose or pyruvate. The entry of CH3NH3+ supported by glucose oxidation in an F1F0-ATPase-deficient mutant was blocked by uncoupler. Transport by wild-type cells under similar conditions was significantly inhibited by arsenate. Thus, CH3NH3+ uptake requires both ATP and an electrochemical H+ gradient. This transport activity was lost upon exposure of E. coli to osmotic shock, but could be recovered by incubation of shocked cells with boiled shock fluid or with glucose plus K+ in the presence of chloramphenicol. Similar reconstitution was observed in K+-depleted parental strains, but not in a mutant defective in K+ transport, demonstrating a requirement for internal K+. However, external K+ proved to be a noncompetitive inhibitor (Ki = 1 mM) of CH3NH3+ uptake by K+ -replete bacteria. External Na+ had no effect on transport. The addition of NH4+ or CH3NH3+ induced a rapid exodus of intracellular 86Rb+, an analog which was able to substitute for K+. The molar ratio of CH3NH3+ uptake to Rb+ exit was 1.12 +/- 0.11. These findings support a mechanism for CH3NH3+ (NH4+) accumulation which requires K+ antiport (exchange) and is driven by the electrochemical K+ gradient.
D017920:Antiporters; D001120:Arginine / Q000378:metabolism; D001692:Biological Transport; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000378:metabolism*; D027682:Cation Transport Proteins; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008744:Methylamines / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D011392:Proline / Q000378:metabolism
D017920:Antiporters; D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D008565:Membrane Proteins; D008744:Methylamines; C043130:ammonium-potassium antiporter; D001120:Arginine; D011392:Proline; C027451:methylamine; D011188:Potassium