Partial purification and properties of a cyclic 3',5'-AMP-independent protein kinase from rat liver.

1. A cyclic 3',5'-AMP-independent protein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) from rat liver cytosol was partially purified and characterized. Purification by (NH4)2SO4 precipitation, DEAE-cellulose, Bio Gel A-0.5 m and cellulose phosphate chromatography increased the specific activity about 700-fold. 2. An endogenous protein substrate was closely associated with the protein kinase and was not separable from this enzyme up to the cellulose phosphate stage. After phosphorylation, chromatography with Bio Gel A-0.5 m partially separated this endogenous phosphoprotein from the enzyme activity; this dissociation had no apparent effect on kinase activity with casein or phosvitin as substrates, or on the apparent molecular weight of the enzyme (approx. 158,000). 3. This protein kinase with casein, phosvitin, or the endogenous substrate was totally insensitive to the thiol reagents, p-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), iodoacetamide, and N-ethylmaleimide. The enzyme was also unaffected by cyclic 3',5'-AMP, heat-stable protein kinase inhibitor, and the regulatory subunit of a cyclic 3',5'-AMP-dependent protein kinase.