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 identified as plasma membrane Ca2+-ATPase by its interaction with calmodulin and monoclonal antibodies produced against red blood cell (RBC) Ca2+-ATPase, and by thapsigargin insensitivity. The purpose of the study was to establish whether two regulators of the RBC Ca2+-ATPase, calmodulin and protein kinase C (PKC), affect the Ca2+-ATPase isolated from excitable cells and whether their effects are comparable to those on the RBC Ca2+-ATPase. We found that calmodulin and PKC activated both enzymes. There were significant quantitative differences in the phosphorylation 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 rate of PKC catalyzed phosphorylation (in the presence of 12-myristate 13-acetate phorbol) was approximately two times slower for SM enzyme. While phosphorylation of RBC Ca2+-ATPase approached maximum level at around 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 associated with diversities in Ca2+-ATPase function in erythrocytes and neuronal cells and different isoforms composition.
Kosk-Kosicka
D
D
Department of Anesthesiology/CCM, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-4163, USA.
Zylińska
L
L
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology*; D002462:Cell Membrane / Q000201:enzymology*; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D004912:Erythrocytes / Q000166:cytology / Q000201:enzymology*; D009474:Neurons / Q000166:cytology / Q000201:enzymology*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013570:Synaptic Membranes / Q000201:enzymology; D019284:Thapsigargin / Q000494:pharmacology
D002147:Calmodulin; D019284:Thapsigargin; D011493:Protein Kinase C; D000252:Calcium-Transporting ATPases