The role of the sites for ATP of the Ca2+-ATPase from human red cell membranes during Ca2+-phosphatase activity

1. In the presence of ATP, the Ca2+ pump of human red cell membranes catalyzes the hydrolysis of p-nitrophenyl phosphate. The requirement for ATP of the Ca2+-p-nitrophenylphosphatase activity was studied in relation to the two classes of site for ATP that are apparent during Ca2+ -ATPase activity. 2. (a) The K0.5 for ATP as activator of the Ca2+ -p-nitrophenylphosphatase extrapolated at 0 mM PNPP is equal to the Km of the Ca2+ -ATPase. (b) PNPP competes with ATP and its effectiveness is the same regardless the nucleotide acts as the substrate of the Ca2+ -ATPase or as activator of the Ca2+ -p-nitrophenylphosphatase. 3. PNPP at the high-affinity site does not substitute for ATP as activator of the Ca2+ -p-nitrophenylphosphatase. 4. At ATP concentrations that almost saturate the high-affinity site, Ca2+ -p-nitrophenylphosphatase activity increases as a function of PNPP along an S-shaped curve, while Ca2+ -ATPase activity is partially inhibited along a curve of the same shape and apparent affinity. The fraction of Ca2+ -ATPase activity which is inhibited by PNPP is that which results from occupation of the low-affinity site by ATP. 5. Activation of the Ca2+ -ATPase by ATP at the low-affinity site is associated with inhibition of the Ca2+ -p-nitrophenylphosphatase activity. Both phenomena take place with the same apparent affinity and along curves of the same shape. 6. Experimental results suggest that: (a) the Ca2+ -p-nitrophenylphosphatase activity depends on ATP at the high-affinity site; (b) PNPP is hydrolyzed at the low-affinity site; (c) Ca2+ -ATPase activity at the high-affinity size persists during Ca2+ -p-nitrophenylphosphatase activity.