REGULATION OF THE ERYTHROCYTE CA2-ATPASE AT HIGH PH()

The activation of the Ca2+-ATPase from erythrocyte membranes at high p H has been investigated. Following alkalinization and in the absence o f regulators, the enzyme exhibits a very high affinity for Ca2+ and a decreased maximal velocity. Either addition of calmodulin, addition of acidic phospholipids, or controlled trypsinization decreases the conc entration of effector required to elicit half-maximal activation of th e enzyme for calcium to similar values. The increase in affinity for C a2+, however, is smaller than that observed at neutral pH. The maximal velocity at high pH becomes insensitive to both calmodulin and contro lled proteolysis, although calmodulin binds to the protein with simila r affinities at pH 7.0 and 8.0, as indicated by similarity in binding to a calmodulin-Sepharose resin and in dependence on calmodulin concen trations when the pH is increased. In contrast to the attenuated effec ts of calmodulin and proteolysis, at pH 8.0 the enzyme is susceptible to stimulation by phospholipids, indicating that the pathway for trans duction of the signal from phospholipids is distinct from that pathway engaged by calmodulin and/or trypsinization. At pH 8.0, phosphatidyli nositol induces the modulatory effect of ATP at the regulatory site bu t calmodulin does not. We suggest that the intraenzymic connection bet ween the calmodulin-binding, autoinhibitory peptide and the nucleotide domain of the enzyme is impaired upon alkalinization, which would acc ount for the differing abilities of the activators to modulate the ATP effects.