Metallo-enzymes Released from Escherichia coli by Osmotic Shock

Escherichia coli cells were grown in the presence of radioactive 65Zn and subjected to osmotic shock. Two enzymes, 5'-nucleotidase and cyclic phosphodiesterase, were purified from the shock fluid. Other evidence indicates that 5'-nucleotidase was brought to a stage of purification corresponding to that of a homogeneous protein. The purifications were accompanied by a considerable enrichment with respect to 65Zn, which could not be removed by dialysis. Superimposable peaks of enzyme activity and 65Zn were observed for both enzymes. Cyclic phosphodiesterase and 5'-nucleotidase were inactivated after prolonged exposure to ethylenediaminetetraacetate and partial reactivation was achieved with Zn++. Treatment of 5'-nucleotidase with acid caused loss of activity associated with release of 65Zn. The preparation could be reactivated by low concentrations of each of several divalent metal ions. It is suggested that cyclic phosphodiesterase and 5'-nucleotidase are metalloproteins and possibly zinc metalloenzymes.