Two Ca2+-requiring p-nitrophenylphosphatase activities of the highly purified Ca2+-pumping adenosinetriphosphatase of human erythrocyte membranes, one requiring calmodulin and the other ATP.
The highly purified Ca2+-pumping ATPase from human erythrocyte membranes displays two p-nitrophenylphosphatase (NPPase) activities: one of these requires calmodulin and low concentrations of Ca2+, while the other requires ATP and higher Ca2+ concentrations. The free Ca2+ concentrations required for the expression of the two NPPase activities differed very substantially. Both activities required high free Mg2+ concentrations and displayed simple hyperbolic kinetics toward p-nitrophenyl phosphate (NPP) with a Km in the range of 5-20 mM. Study of the dependence of the calmodulin-stimulated NPPase on Mg2+ and NPP indicated that the Mg-NPP complex is not the substrate of the enzyme. Under conditions optimal for ATP-requiring NPPase (1 mM free Ca2+), the Ca2+-ATPase displayed simple hyperbolic kinetics with a low Km for ATP. NPP competitively inhibited this activity, and the apparent Ki for NPP was less than 1 mM, much lower than the Km for NPP as a substrate. If NPP were inhibiting the ATPase by binding at the same site at which NPP is hydrolyzed, the apparent Ki for NPP as inhibitor would be the same as the Km for NPP as substrate. (Under these circumstances, the apparent Ki and the Km can be directly compared, since NPP was being hydrolyzed under both circumstances.) Since Ki was much lower than Km, NPP must have been inhibiting at another site; thus, these data show the existence of two types of NPP sites on the enzyme, one at which NPP is hydrolyzed and the other at which it inhibits ATP hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
D009597:4-Nitrophenylphosphatase / Q000097:blood*; D000255:Adenosine Triphosphate / Q000097:blood*; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000302:isolation & purification; D002147:Calmodulin / Q000097:blood*; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D010744:Phosphoric Monoester Hydrolases / Q000097:blood*
D002147:Calmodulin; D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D009597:4-Nitrophenylphosphatase; D000252:Calcium-Transporting ATPases; D005033:Ethylmaleimide; D002118:Calcium