Regulation of the erythrocyte Ca(2+)-ATPase at high pH.
The activation of the Ca(2+)-ATPase from erythrocyte membranes at high pH has been investigated. Following alkalinization and in the absence of 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 concentration of effector required to elicit half-maximal activation of the enzyme for calcium to similar values. The increase in affinity for Ca2+, however, is smaller than that observed at neutral pH. The maximal velocity at high pH becomes insensitive to both calmodulin and controlled proteolysis, although calmodulin binds to the protein with similar affinities at pH 7.0 and 8.0, as indicated by similarity in binding to a calmodulin-Sepharose resin and in dependence on calmodulin concentrations when the pH is increased. In contrast to the attenuated effects of calmodulin and proteolysis, at pH 8.0 the enzyme is susceptible to stimulation by phospholipids, indicating that the pathway for transduction of the signal from phospholipids is distinct from that pathway engaged by calmodulin and/or trypsinization. At pH 8.0, phosphatidylinositol induces the modulatory effect of ATP at the regulatory site but calmodulin does not. We suggest that the intraenzymic connection between the calmodulin-binding, autoinhibitory peptide and the nucleotide domain of the enzyme is impaired upon alkalinization, which would account for the differing abilities of the activators to modulate the ATP effects.
de Carvalho-Alves
P C
PC
Departamento de Bioquímica Médica, ICB, CCS, Universidade Federal de Rio de Janeiro, Brasil.
Freire
M M
MM
Barrabin
H
H
Scofano
H M
HM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000097:blood; D000252:Calcium-Transporting ATPases / Q000097:blood*; D002147:Calmodulin / Q000097:blood*; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010743:Phospholipids / Q000494:pharmacology; D013552:Swine; D014357:Trypsin / Q000494:pharmacology; D003189:p-Methoxy-N-methylphenethylamine / Q000494:pharmacology
D002147:Calmodulin; D010743:Phospholipids; D003189:p-Methoxy-N-methylphenethylamine; D000255:Adenosine Triphosphate; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D002118:Calcium