The activation of Phosphatase Activity of the Ca2+-ATPase from Human Red Cell Membranes by Calmodulin, ATP and Partial Proteolysis

65. ROSSI J.P.F.C, GARRAHAN, P.J. Y REGA, A.F.

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 1986 vol. 858 p. 21 - 30

0005-2736

(1) Depending on the assay conditions, the ability of the Ca2+-ATPase from intact human red cellmembranes to catalyze the hydrolysis of p-nitrophenyiphosphate is elicited by either calmodulin or ATP.The response of the phosphatase activity to p-nitrophenylphosphate, ATP, Mg 2+ and K + is the same for theactivities elicited by ATP or by caimodulin, suggesting that a single process is responsible for both activities.(2) In media with calmodulin, high-affinity activation is followed by high-affinity inhibition of the phosphataseby Ca 2+ so that the activity becomes negligible above 30/tM Ca z+. Under these conditions, additionof ATP leads to a large decrease in the apparent affinity for inhibition by Ca 2+. (3) In membranes submittedto partial proteolysis with trypsin, neither calmodulin nor Ca 2+ are needed and phosphatase activity ismaximal in media without Ca 2+. This is the first report of an activity sustained by the Ca2+-ATPase of redcell membranes in the absence of Ca 2+. Under these conditions, however, ATP still protects againsthigh-affinity inhibition by Ca 2+. These results strongly suggest that during activation by calmodulin, Ca 2+ isneeded only to form the calmodulin-Ca 2 + complex which is the effective cofactor. (4) Protection by ATP ofthe inhibitory effects of Ca 2+ and the induction of phosphatase activity by ATP + Ca 2+ suggests thatactivation of the phosphatase by Ca 2 + in media with ATP requires the combination of the cation at sites inthe ATPase. (5) Results can be rationalized assuming that E2, the conformer of the Ca2+-ATPase, isendowed with phosphatase activity. Under this assumption, either the calmodulin-Ca 2+ complex or partialproteolysis would elicit phosphatase activity by displacing the equilibrium between E I and E 2 towards E 2.On the other hand, ATP + Ca 2+ would elicit the activity by establishing through a phosphorylation-dephosphorylationcycle a steady-state in which E 2 predominates over other conformers of the ATPase.