Purification and reconstitution of a Ca2+ pump from human platelets.

A Ca2+-ATPase from human platelets has been purified after solubilization with octyl glucoside. Following chromatography on Sepharose 4B and hydroxylapatite the Ca2+-ATPase had a specific activity of 1.1 mumol of ATP hydrolyzed/min/mg of protein at 30 degrees C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that 80% of the preparation was a polypeptide with a molecular weight of approximately 100,000. The major contaminant had a molecular weight of 89,000, and both proteins cross-reacted with anti-serum against the Ca2+-ATPase from rabbit skeletal muscle sarcoplasmic reticulum. It is likely that the 89,000-dalton polypeptide is an inactive proteolysis product of the Ca2+-ATPase. The kinetic properties of the purified ATPase with Ca2+ and MgATP were quite similar to those of the sarcoplasmic reticulum ATPase. Ca2+ transport activity was reconstituted by dialysis of the octyl glucoside. The platelet Ca2+ pump transported 2 Ca2+ for each ATP molecule hydrolyzed. Thus the platelet Ca2+ pump is similar to the ATPase from the sarcoplasmic reticulum in structure and function. Furthermore, the Ca2+ pump is a major membrane component in platelet membranes, and this emphasizes the importance of Ca2+ fluxes in platelet function.