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Impairment and Restoration of the Energized State in Membrane Vesicles of a Mutant of Escherichia coli Lacking Adenosine Triphosphatase

From the ¹ From the Division of Research, National Jewish Hospital and Research Center, Denver, Colorado 80206, and The Department of Microbiology, University of Colorado School of Medicine, Denver, Colorado 80220
² From the Department of Biological Sciences, Stanford University, Stanford, California 94305

We have studied a mutant of Escherichia coli, DL-54, which lacks Ca²⁺, Mg²⁺-stimulated ATPase activity and cannot use ATP as an energy source for active transport and other work functions. The purpose of the investigation was to explain the unexpected observation that vesicles of this mutant were also defective in coupling respiration to nutrient accumulation.

In vesicles of mutant DL-54, unlike those of the wild type, oxidation of d-lactate did not support accumulation of amino acids, rubidium (in presence of valinomycin), or the lipid-soluble dibenzyldimethylammonium ion. All these functions were restored by treating the vesicles with N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the ATPase complex. Accumulation of amino acids in response to an artificial electrical potential (imposed by valinomycin-mediated K⁺ efflux) was also deficient in the mutant but was restored by DCCD. We infer that DCCD restores the capacity of mutant vesicles to establish an electrical potential across the membrane which, in turn, may be the immediate driving force for active transport and other work functions.

Extrusion of protons by the respiratory chain is a possible mechanism for the generation of a membrane potential. Vesicles of DL-54, unlike those of the parent, exhibit no proton pulse unless they have been first treated with DCCD. A plausible basis for all these phenomena was found in the observation that the permeability of the membrane to protons was far higher in DL-54 than in the parent strain. Treatment with DCCD reduced the proton permeability to the low level characteristic of the wild type and thus prevented dissipation of the electrical potential.

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