D-Gluconate transport in Arthrobacter pyridinolis. Metabolic trapping of a protonated solute.
D-Gluconate uptake was studied in whole cells of Arthrobacter pyridinolis; the uptake activity was inducible, mutable and showed saturation kinetics (Km = 5 micrometer). Uptake of D-gluconate was not mediated by a phosphoenol-pyruvate : hexose phosphotransferase system, nor was it directly energized by ATP. A transmembrane pH gradient, delta pH, of --63 mV was generated by A. pyridinolis cells at pH 6.5, while at pH 7.5, delta pH = 0. Addition of 8 micrometer D-gluconate significantly reduced the delta pH. The transmembrane electrical potential, delta psi, which was --87 mV over a range of pH from 5.5 to 7.5, was unaffected by the presence of substrate. D-Gluconate accumulated at the same rate and as the protonated solute, at both pH 6.5 and 7.5. Experiments in which a diffusion potential was generated in cyanide-treated cells, indicated that the delta psi did not energize transport. Rather, the rate of D-gluconate uptake metabolism: (a) treatment of cells with valinomycin or nigericin, under conditions in which there was a loss of intracellular potassium, inhibited both D-gluconate uptake and the metabolism of pre-accumulated D-gluconate; (b) the effects of cyanide and azide on D-gluconate uptake were much more severe at pH 6.5 than pH 7.5, a pattern which paralleled the effects of these inhibitors on D-gluconate metabolism; (c) extraction and chromatography of intracellular label from D-gluconate uptake revealed that accumulation of unaltered D-gluconate was negligible; (d) a series of mutant strains with lower D-gluconate kinase activities also exhibited low rates of D-gluconate uptake; (e) spontaneous revertants of these mutant strains consistently regained both D-gluconate kinase activity and wild type levels of uptake.
D001149:Arsenates / Q000494:pharmacology; D001173:Arthrobacter / Q000187:drug effects / Q000378:metabolism*; D001386:Azides / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002087:Butyrates / Q000378:metabolism; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D003486:Cyanides / Q000494:pharmacology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005942:Gluconates / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009550:Nigericin / Q000494:pharmacology; D014634:Valinomycin / Q000494:pharmacology
D001149:Arsenates; D001386:Azides; D002087:Butyrates; D003486:Cyanides; D005942:Gluconates; D014634:Valinomycin; D004024:Dicyclohexylcarbodiimide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D009550:Nigericin