IRREVERSIBLE EFFECTS OF CALCIUM-IONS ON THE PLASMA-MEMBRANE CALCIUM-PUMP

The calcium pump of human red cells can be irreversibly activated by p reincubation of the membranes in the presence of calcium ions, with a pattern reminiscent of that produced by controlled trypsin attack. Wit h 1 mM Ca2+, the activity of the basal enzyme increases three to fourf old over 30 to 60 min, to levels about half those obtained in the pres ence of calmodulin. On the whole, the effect occurs slowly, with a ver y low Ca2+ affinity at 37 degrees C and is unaffected by serine-protea se inhibitors. The activation caused by 1 mM Ca2+ is little affected b y leupeptin (a thiol-protease inhibitor) and that obtained at 10 mu M Ca2+ is not inhibited. Preincubations at 0 degrees C also lead to acti vation, to a level up to half that seen at 37 degrees C, and the effec t is not affected by leupeptin or antipain. No activation is observed by preincubating soluble purified Ca,Mg-ATPase in Ca2+-containing solu tions at 37 degrees C. Instead, calcium ions protect the detergent-sol ubilized enzyme from thermal inactivation, the effect being half-maxim al between 10 and 20 mu M Ca2+. We conclude that the activation of the membrane-bound Ca,Mg-ATPase by Ca2+ should result from an irreversibl e conformational change in the enzyme and not from attack by a membran e-bound protease, and that this change presumably arises from the rele ase of inhibitory particles existing in the original membrane preparat ions.