Activation potassium efflux from Escherichia coli by glutathione metabolites

Abstract

The mechanism by which N-ethylmaleimide (NEM) elicits potassium efflux from Escherichia coli has been investigated. The critical factor is the formation of specific glutathione metabolites that activate transport systems encoded by the kefB and kefC gene products. Formation of N-ethyl-succinimido-S-glutathione (ESG) leads to the activation of potassium efflux via these transport systems. The addition of dithiothreitol and other reducing agents to cells reverses this process by causing the breakdown of ESG and thus removing the activator of the systems. Chlorodinitrobenzene, p-chloromercuribenzoate and phenylmaleimide provoke similar effects to NEM. lodoacetate, which leads to the formation of S-carboxymethyl-glutathione, does not activate the systems but does prevent the action of NEM. It is concluded that the KefB and KefC systems are gated by glutathione metabolites and that the degree to which they are activated is dependent upon the nature of the substituent on the sulphydryl group.

The distribution of homologues of the Escherichia coli KefC K+-efflux system in other bacterial species

“…We have previously shown that the involvement of glutathione in the NEMactivation of K+ efflux can be demonstrated by converting the glutathione pool to S-carboxymethylglutathione by treatment with iodoacetate. S-Carboxymethylglutathione is not a strong activator of the E. coli KefC system and subsequent incubation with NEM does not elicit K+ efflux because there is no free glutathione to react with NEM and form ESG (Elmore et al, 1990). Re-incubation of iodoacetate-treated E. coli cells with glutathione leads to the re-establishment of the cytoplasmic glutathione pool and restoration of sensitivity of K+ efflux to NEM (Elmore et al, 1990).…”

“…The activation of the KefC system can be achieved by the reaction of N-ethylmaleimide (NEM) with cellular glutathione to form the activator N-ethylsuccinimido-S-glutathione (ESG) (Elmore et al, 1990). We therefore sought to elicit K+ efflux from a range of bacteria by the addition of NEM (0.5 mM final concentration) to cells washed and suspended in K+-free medium (Fig.…”

“…S-Carboxymethylglutathione is not a strong activator of the E. coli KefC system and subsequent incubation with NEM does not elicit K+ efflux because there is no free glutathione to react with NEM and form ESG (Elmore et al, 1990). Re-incubation of iodoacetate-treated E. coli cells with glutathione leads to the re-establishment of the cytoplasmic glutathione pool and restoration of sensitivity of K+ efflux to NEM (Elmore et al, 1990). We therefore sought to apply this protocol to the above group of organisms.…”

“…This reversal arises from the breakdown of ESG to release glutathione (Elmore et al, 1990) and from the release of glutathione from cellular dithiols (A. J . Lamb & I. R. Booth, unpublished data).…”

“…The presence of glutathione in each of the species was investigated by assaying for the formation of S-carboxymethylglutathione when cells were incubated with iodoacetate. Metabolites were extracted from cells incubated with iodo[2-14C]acetate [ 1 mM final concentration; 54 pCi pmol-l (2 MBq pmol-l) specific activity] with trichloroacetic acid and subjected to chromatography on PEI-cellulose plates (Elmore et al, 1990). In each case a single major metabolite which had the same RF value as S-carboxymethylglutathione (Elmore et al, 1990) was detected by autoradiography and the amount of the metabolite was determined ( Table 1).…”