The activation of phosphatase activity of the Ca2+-ATPase from human red cell membranes by calmodulin, ATP and partial proteolysis.
Depending on the assay conditions, the ability of the Ca2+-ATPase from intact human red cell membranes to catalyze the hydrolysis of p-nitrophenylphosphate is elicited by either calmodulin or ATP. The response of the phosphatase activity to p-nitrophenylphosphate, ATP, Mg2+ and K+ is the same for the activities elicited by ATP or by calmodulin, suggesting that a single process is responsible for both activities. In media with calmodulin, high-affinity activation is followed by high-affinity inhibition of the phosphatase by Ca2+ so that the activity becomes negligible above 30 microM Ca2+. Under these conditions, addition of ATP leads to a large decrease in the apparent affinity for inhibition by Ca2+. In membranes submitted to partial proteolysis with trypsin, neither calmodulin nor Ca2+ are needed and phosphatase activity is maximal in media without Ca2+. This is the first report of an activity sustained by the Ca2+-ATPase of red cell membranes in the absence of Ca2+. Under these conditions, however, ATP still protects against high-affinity inhibition by Ca2+. These results strongly suggest that during activation by calmodulin, Ca2+ is needed only to form the calmodulin-Ca2+ complex which is the effective cofactor. Protection by ATP of the inhibitory effects of Ca2+ and the induction of phosphatase activity by ATP + Ca2+ suggests that activation of the phosphatase by Ca2+ in media with ATP requires the combination of the cation at sites in the ATPase. Results can be rationalized assuming that E2, the conformer of the Ca2+-ATPase, is endowed with phosphatase activity. Under this assumption, either the calmodulin-Ca2+ complex or partial proteolysis would elicit phosphatase activity by displacing the equilibrium between E1 and E2 towards E2. On the other hand, ATP + Ca2+ would elicit the activity by establishing through a phosphorylation-dephosphorylation cycle a steady-state in which E2 predominates over other conformers of the ATPase.
D000255:Adenosine Triphosphate / Q000097:blood*; D002118:Calcium / Q000097:blood; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000097:blood*; D002147:Calmodulin / Q000097:blood*; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000097:blood*; D011188:Potassium / Q000378:metabolism; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
D002147:Calmodulin; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D011188:Potassium; D002118:Calcium