Acetylation with succinimidyl acetate affects both the catalytic site and the regulation of the erythrocyte Ca2+ pump.
Acetylation of lysine residues of the erythrocyte Ca2+ pump using succinimidyl acetate (SA) led to its complete inactivation. In the absence of any of the major activators of the pump (namely calmodulin and acidic phospholipids), ATP fully protected the pump from inactivation by SA, with a K0.5 of 13 microM. This value is very close to the Km of the high-affinity site for ATP, thus suggesting that the residue(s) involved is(are) near or at the catalytic site of the Ca(2+)-ATPase. Furthermore, the presence of 500 microM ATP prevented the acetylation of about two residues per molecule of enzyme. Acetylation by SA also prevented the activation of the Ca2+ pump by calmodulin, acidic phospholipids or controlled trypsin proteolysis. This effect of SA treatment was not avoided by the presence of ATP in the preincubation medium, indicating a second set of modified residues. The fact that the three modes of activation were cancelled in a similar fashion by SA suggests that, although acting via different mechanisms, they share at least a common step in which SA-sensitive lysine residues may participate. Moreover, modification of the pump by SA plus ATP decreased the KCa when the activity was measured in both the absence and presence of calmodulin, suggesting that the residue(s) modified in this case is(are) involved directly in the regulation of the affinity for Ca2+.
Donnet
C
C
IQUIFIB, Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
Caride
A J
AJ
Fernández
H N
HN
Rossi
J P
JP
D000107:Acetylation; D000328:Adult; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010743:Phospholipids / Q000378:metabolism; D013388:Succinimides / Q000378:metabolism*
D002147:Calmodulin; D010743:Phospholipids; D013388:Succinimides; C088964:succinimidyl acetate; D000252:Calcium-Transporting ATPases