Activation of erythrocyte Ca2+-plus-Mg2+-stimulated adenosine triphosphatase by protein kinase (cyclic AMP-dependent) inhibitor. Comparison with calmodulin

Abstract

Purified protein kinase (cyclic AMP-dependent) inhibitor (PKI) from bovine heart stimulated Ca2++Mg2+-stimulated ATPase activity in human erythrocytes, the stimulation being maximal at 2μg/0.6ml. By contrast, PKI from rabbit skeletal muscle had no effect. Bovine heart PKI stimulated Ca2++Mg2+-stimulated ATPase by increasing the Ca2+-sensitivity of the enzyme. This contrasted with the stimulation by calmodulin, which increased the maximum velocity of the Ca2++Mg2+-dependent ATPase in addition to its effect on the Ca2+-sensitivity. Both membrane-bound and Triton X-100-solubilized Ca2++Mg2+-stimulated ATPase activities were stimulated by PKI, indicating that the stimulation did not require an intact membrane structure. At low Ca2+ concentration the stimulation by PKI and saturating concentrations of calmodulin were additive, suggesting that the two effectors acted by distinct mechanisms. Although 5μm-cyclic AMP inhibited Ca2++Mg2+-stimulated ATPase activity by about 20% when measured at low ATP concentrations, probably by stimulation of phosphorylation by an endogenous protein kinase, the stimulation by PKI (about 100%) was not solely due to its antagonism of the protein kinase. This interpretation was supported by a number of observations. First, modification of arginine residues of bovine heart PKI abolished its inhibition of cyclic AMP-dependent protein kinase, but had no effect on the stimulation of Ca2++Mg2+-stimulated ATPase. Secondly, trifluoperazine (20μm) antagonized the stimulation of Ca2++Mg2+-dependent ATPase by PKI, similarly to its antagonism of calmodulin stimulation, but it did not affect the inhibition of protein kinase by PKI. We conclude that different mechanisms are involved in the inhibition of protein kinase and the stimulation of Ca2++Mg2+-stimulated ATPase by PKI.