A Major Cell Envelope Protein Of Escherichia Coli K12: Purification And Properties
The isolation and characterization of a major cell envelope protein from E. coli K12, the so-called "succinate eluted protein" (SEP) is reported. SEP was released by EDTA-sucrose and osmotic shock treatments, or during the formation of spheroplasts. The protein was purified on columns of aspartate-Sepharose (A-S) an affinity matrix previously used for the isolation of dicarboxylate transport components. SEP was the major protein eluted when the A-S columns were equilibrated in low ionic strength buffer (10 mM phosphate containing 5 mM EDTA). SEP was purified to apparent homogeneity as judged by SDS-PAGE, IEF and HPLC/gel filtration. SEP stimulated succinate transport in whole cells when added externally. The binding of ({dollar}sp{lcub}35{rcub}{dollar}S) -SEP to whole cells was examined. SEP was bound to whole cells with a strong affinity (K{dollar}sb{lcub}rm D{rcub}{dollar} = 30 nM). The binding was dependent on the presence of porin and normal K12 LPS. Mutants that lacked porin, or had shortened LPS were unable to bind SEP or transport succinate. SEP binding to porin immobilized on Sepharose was also demonstrated. This binding was very specific in the presence of low concentrations of SDS and was fairly resistant to disruption by NaCl. Binding of SEP to whole cells or porin-Sepharose was not affected by dicarboxylic acids. Increased succinate transport activity and increased levels of SEP occurred when cells were grown in minimal media. In minimal media the 56K SEP comprised 20-25% of the proteins released by osmotic shock. Further studies indicated that the interaction of SEP with A-S was not that strong as the protein could be eluted with 30 mM phosphate, differentiating it from a previously characterized 16K dicarboxylic acid binding protein which was retained by A-S equilibrated in 50 mM phosphate and 50 mM arsenate. Elution with 30 mM phosphate was used to isolate increased amounts of SEP free of other proteins or succinate. Pure SEP had no detectable succinate binding activity. The mechanism of action of SEP in stimulating the transport of succinate may involve the blocking of charged groups close to the opening of the porin channel.
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