Abstract
The use of novel synthetic peptides to measure peptide transport by spectrophotometric means is described. These peptides contain glycine residues alpha-substituted with thiophenol and are recognized as substrates by both peptide transport systems and intracellular peptidases of Escherichia coli (Kingsbury et al., Gilvarg, C., Proc. Natl. Acad. Sci. U.S.A. 81:4573-4576, 1984). Transport and peptidase cleavage results in the intracellular release of thiophenol, which exits rapidly from the cell. The release of thiophenol from these peptides by cell suspensions can be measured with Ellman sulfhydryl reagent [5,5'-dithiobis(2-nitrobenzoic acid)] and provides a direct determination of the rate of peptide transport. The reductions in thiophenol release from these peptides resulting from the addition of peptide competitors enable the affinities of the competitors for their transport systems to be determined. By this method, it is shown that the dipeptide transport system is more restrictive with respect to changes in the amino acid sidechains of its substrates than those of the oligopeptide transport system.