ACETYLATION WITH SUCCINIMIDYL ACETATE AFFECTS BOTH THE CATALYTIC SITEAND THE REGULATION OF THE ERYTHROCYTE CA2+ PUMP

Acetylation of lysine residues of the erythrocyte Ca2+ pump using succ inimidyl acetate (SA) led to its complete inactivation. In the absence of any of the major activators of the pump (namely calmodulin and aci dic phospholipids), ATP fully protected the pump from inactivation by SA, with a K-0.5 of 13 mu M. This value is very close to the K-m of th e high-affinity site for ATP, thus suggesting that the residue(s) invo lved is(are) near or at the catalytic site of the Ca2+-ATPase. Further more, the presence of 500 mu M 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 a voided by the presence of ATP in the preincubation medium, indicating a second set of modified residues. The fact that the three modes of ac tivation were cancelled in a similar fashion by SA suggests that, alth ough acting via different mechanisms, they share at lease a common ste p in which SA-sensitive lysine residues may participate. Moreover, mod ification of the pump by SA plus ATP decreased the K-Ca when the activ ity was measured in both the absence and presence of calmodulin, sugge sting that the residue(s) modified in this case is(are) involved direc tly in the regulation of the affinity for Ca2+.