Peptidases in Spheroplasts of Escherichia coli K-12

From the ¹ From the Department of Biochemistry, Yale University, New Haven, Connecticut 06510

To determine whether enzymes that catalyze the hydrolysis of dipeptides are located at or near the surface of Escherichia coli strain K-12, the bacteria were converted to Spheroplasts by treatment with ethylenediaminetetraacetate and lysozyme, and the enzymic activity of the medium surrounding the spheroplasts and of the spheroplast lysate was compared with the activity of a total cell lysate. The substrates were glycyl-L-leucine, glycyl-L-phenylalanine, L-leucylglycine, and L-phenylalanylglycine. Under a variety of experimental conditions, peptidase assays showed that essentially all the peptidase activity of lysed cells is retained within the spheroplasts; the relative amount of enzymic activity toward each dipeptide that is released into the surrounding medium as a result of spheroplast formation is almost identical with the extent of ß-galactosidase leakage.

A significant portion of the peptidase activity shown by cell lysates exists in a latent condition in intact cells, which show low, but equal, specific activity toward all the peptides. In contrast, cell lysates and sonic extracts and spheroplast lysates exhibit markedly different hydrolytic activity toward the four substrates. A variation in specific activity toward the substrates also is evident after treatment of intact cells with toluene, but toluene treatment permits the expression of only a portion of the activity that is latent in the intact cells and is evident in cell lysates or spheroplast lysates.

The activity characteristic of each type of bacterial preparation under a specific set of assay conditions is approximately the same for preparations made from cells harvested from cultures during exponential growth or shortly after the attainment of maximal growth. However, the specific activity of a given bacterial preparation to a given peptide varies depending on the concentration of EDTA and of Mn²⁺ in the assay mixture. When the concentrations of these substances are varied, it is possible to distinguish readily between enzymes that act on leucylglycine, on phenylalanylglycine, and on the two dipeptides with an amino-terminal glycine residue.

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