Adenosine 3′,5′-Monophosphate-Independent Protein Kinase Activities in the Bovine Adrenal Cortex Cytosol*

Abstract

The cAMP-independent protein kinase activity previously described in the bovine adrenal cortex cytosol was resolved into four active moieties by phosphocellulose chromatography. The four isolated protein kinases exhibited the highest activity with casein as substrate and were not influenced by the presence of various cyclic nucleotides or by the heat-stable inhibitory protein of Ashby and Walsh. According to their functional as well as some physicochemical properties, the four isolated active moieties may be classified into two groups termed A (PC 1) and G (PC 2, 3, and 4) casein kinase (CK) types, respectively. Casein kinase PC 1 had the lowest affinity for phosphocellulose, was stimulated about 10-fold in the presence of 100 mm MgCl₂, could use ATP (Km = 15 μm) but not GTP as phosphoryl donor, was not significantly influenced by the type II heat-stable inhibitor of Szmigielski et al., and sedimented at 2.5S_20.W in a 0.5- m NaCl sucrose density gradient. By contrast, PC 2, 3, and 4 casein kinases were retained on phosphocellulose, were 50- to 100-fold stimulated upon the addition of 100 mm MgCl₂ in the assay, used GTP (Km = 10–20 μm) as well as ATP (Km = 7.1– 8.2 μm) as a source of phosphate, and were sensitive to the heatstable type II inhibitor. The three enzymatic activities similarly sedimented at 5.5S_2o,w in a sucrose density gradient. Hemin had no significant effect on the isolated casein kinase activities; polyamines at millunolar concentrations appeared to be powerful activators of the PG 2, 3, and 4 enzyme class, whereas PC 1 was less sensitive to these compounds. The four isolated protein kinases incorporated phosphate from ATP (PC 1) or ATP and GTP (PC 2, 3, and 4) into endogenous material; this might reflect an autophosphorylation of the enzymatic moieties or the presence of associated endogenous substrate(s) which copurified upon phosphocellulose chromatography. The casein kinases were characterized in the crude adrenal cortex cytosol as two classes, CK-A and CK-G, respectively, on the basis of their functional properties and their sedimentation velocities in a sucrose density gradient. This study has provided a basis to probe the soluble cAMP-independent protein kinase activities in adrenal cortex extracts and thus to delineate their possible implication in relation to the action of effectors of adrenocortical functions. (Endocrinology106: 750, 1980)