PROTEIN-KINASE-C AND CALMODULIN EFFECTS ON THE PLASMA-MEMBRANE CA2-ATPASE FROM EXCITABLE AND NONEXCITABLE CELLS()

We have purified Ca2+-ATPase from synaptosomal membranes (SM)(1) from rat cerebellum by calmodulin affinity chromatography. The enzyme was i dentified as plasma membrane Ca2+-ATPase by its interaction with calmo dulin and monoclonal antibodies produced against red blood cell (RBC) Ca2+-ATPase, and by thapsigargin insensitivity. The purpose of the stu dy was to establish whether two regulators of the RBC Ca2+-ATPase, cal modulin and protein kinase C (PKC), affect the Ca2+-ATPase isolated fr om excitable cells and whether their effects are comparable to those o n the RBC Ca2+-ATPase. We found that calmodulin and PKC activated both enzymes. There were significant quantitative differences in the phosp horylation and activation of the SM versus RBC Ca2+-ATPase. The steady -state Ca2+-ATPase activity of SM Ca2+-ATPase was approximately 3 fold lower and significantly less stimulated by calmodulin. The initial ra te of PKC catalyzed phosphorylation (in the presence of 12-myristate 1 3-acetate phorbol) was approximately two times slower for SM enzyme. W hile phosphorylation of RBC Ca2+-ATPase approached maximum level at ar ound 5 min, comparable level of phosphorylation of SM Ca2+-ATPase was observed only after 30 min. The PKC-catalyzed phosphorylation resulted in a statistically significant increase in Ca2+-ATPase activity of up to 20-40%, higher in the SM Ca2+-ATPase. The differences may be assoc iated with diversities in Ca2+-ATPase function in erythrocytes and neu ronal cells and different isoforms composition.