CHEMICAL MODIFICATION REVEALS INVOLVEMENT OF DIFFERENT SITES FOR NUCLEOTIDE ANALOGS IN THE PHOSPHATASE-ACTIVITY OF THE RED-CELL CALCIUM-PUMP

The calcium pump of plasma membranes catalyzes the hydrolysis of ATP a nd phosphoric esters like p-nitrophenyl phosphate (pNPP). The latter a ctivity requires the presence of ATP and/or calmodulin, and Ca2+ [22, 25], We have studied the effects of nucleotide-analogues and chemical modifications of nucleotide binding sites on Ca2+-pNPPase activity. Tr eatment with fluorescein isothiocyanate (FITC), abolished Ca2+-ATPase and ATP-dependent pNPPase, but affected only 45% of the calmodulin-dep endent pNPPase activity. The nucleotide analogue eosin-Y had an inhibi tory effect on calmodulin-dependent pNPPase (Ki(eosin-Y) = 2 mu M). FI TC treatment increased Ki(eosin-Y) 15 times. Acetylation of lysine res idues with N-hydroxysuccinimidyl acetate inactivates Ca2+-ATPase by mo difying the catalytic site, and impairs stimulation by modulators by m odifying residues outside this site [9]. Acetylation suppressed the AT P-dependent pNPPase with biphasic kinetics. ATP or pNPP during acetyla tion cancels the fast component of inactivation. Acetylation inhibited only partially the calmodulin-dependent pNPPase, but neither ATP nor pNPP prevented this inactivation. From these results we conclude: (i) ATP-dependent pNPPase depends on binding of ATP to the catalytic site; (ii) the catalytic site plays no role in calmodulin-dependent pNPPase . The decreased affinity for eosin-Y of the FITC-modified enzyme, sugg ests that the sites for these two molecules are closely related but no t overlapped. Acetimidation of the pump inhibited totally the calmodul in-dependent pNPPase, but only partially the ATP-pNPPase. Since calmod ulin binds to E-1, the E-1 conformation or the E-2 double left right a rrow E-1 transition would be involved during calmodulin-dependent pNPP ase activity.