The Release of Enzymes from Escherichia coli by Osmotic Shock and during the Formation of Spheroplasts

Abstract

When Escherichia coli cells are converted into spheroplasts by means of ethylenediaminetetraacetate and lysozyme (1)) most of the inducible alkaline phosphatase (2, 3) is released into the surrounding sucrose-tris(hydroxymethyl)aminomethane-HCl medium (4, 5), and a large fraction of the “latent” ribonuclease (6-8) and the ribonucleic acid inhibited deoxyribonuclease (9) is also set free (lO).r We have since found that three additional enzymes are liberated: a Co tf-stimulated 5’-nucleotidase, not previously described in E. co& an acid phosphatase; and a cyclic phosphodiesterase (11, 12). A total of 10 other enzymes have been examined and found to remain entirely within the spheroplasts. Various lines of evidence suggest that the enzymes which are released occur at or near the cell surface. We have also developed a new method for releasing most of these enzymes in high yield without greatly impairing the viability of the cells. The procedure involves osmotic shock. E. coli are first suspended in a concentrated solution of sucrose, which does not penetrate the cells, and ethylenediaminetetraacetate is added. Then they are suddenly shifted to a medium of low osmotic strength. This causes the release of the phosphatases and of the cyclic phosphodiesterase in a yield of 70% or more. A preliminary report of this work has appeared (13).