ATPase activity and Ca2+ transport by reconstituted tryptic fragments of the Ca2+ pump of the erythrocyte plasma membrane.
The purified Ca2+ ATPase of the erythrocyte plasma membrane has been submitted to controlled trypsin proteolysis under conditions that favor either its (putative) E1 or E2 configurations. The former configuration has been forced by treating the enzyme with Ca2+-saturated calmodulin, the latter with vanadate and Mg2+. The E1 conformation leads to the accumulation of a polypeptide of Mr 85 KDa which still binds calmodulin, the E2 conformation to the accumulation of one of Mr 81 KDa which does not. Both fragments arise from the hydrolysis of a transient 90 KDa product which has Ca2+-calmodulin dependent ATPase activity, and which retains the ability to pump Ca2+ in reconstituted liposomes. Highly enriched preparations of the 85 and 81 KDa fragments have been obtained and reconstituted into liposomes. The former has limited ATPase and Ca2+ transport ability and is not stimulated by calmodulin. The latter has much higher ATPase and Ca2+ transport activity. It is proposed that the Ca2+ pumping ATPase of erythrocytes plasma membrane contains a 9 KDa domain which is essential for the interaction of the enzyme with calmodulin and for the full expression of the hydrolytic and transport activity. This putative 9 KDa sequence contains a 4 KDa "inhibitory" domain which limits the activity of the ATPase. In the presence of this 4 KDa sequence, i.e., when the enzyme is degraded to the 85 KDa product, calmodulin can still be bound, but no longer stimulates ATPase and Ca2+ transport.
D000255:Adenosine Triphosphate / Q000502:physiology; D001693:Biological Transport, Active; D002118:Calcium / Q000097:blood*; D000252:Calcium-Transporting ATPases / Q000097:blood*; D002147:Calmodulin / Q000097:blood / Q000502:physiology; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D007473:Ion Channels / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D010446:Peptide Fragments / Q000097:blood; D014357:Trypsin
D002147:Calmodulin; D007473:Ion Channels; D008081:Liposomes; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D002118:Calcium