Protein Kinase Activity in Escherichia coli

Abstract

Two ATP-dependent phosphorylating activities in E. coli have been separated by ammonium sulfate fractionation. An enzyme form of low solubility, together with some endogenous substrates, is separated from ribosomes by ultracentrifugation, is eluted from a column of Bio-Gel P-200 in the void volume, and is relatively stable to heat. A divalent cation is required for activity; the rate of endogenous phosphorylation is 4-fold greater with Mn²⁺ than with Mg²⁺. Histones and protamine serve as weak exogenous substrates; purified E. coli ribosomes, phosvitin and casein are not phosphorylated. Cyclic 3’,5’-AMP (1 μM) has no effect on this kinase activity. A somewhat labile cyclic AMP stimulation (6-fold) of endogenous phosphorylation has been observed with a more soluble kinase fraction. (Other enzyme preparations have shown only cyclic AMP-independent activity.) Activity is optimal at pH 7. Mn²⁺ (1 mM) and Mg²⁺ (10 mM) are equally effective in supporting the endogenous phosphorylation. Heterogeneity of the endogenous substrates is indicated by the different subunit molecular weights of the phosphorylated products (about 66,000, 22,000 and 17,000). All three products are hydrolyzed by pronase, but not by RNAase or DNAase, and are dephosphorylated by alkaline phosphatase. The stability of the phosphoryl bond is characteristic of a phosphate ester rather than of an acyl phosphate or a phosphoramidate.