Effects of affinity-purified antibodies on the Ca2+ pumping ATPase of erythrocyte membranes.
Antibodies raised in rabbits against the purified erythrocyte membrane Ca2+ pumping ATPase were affinity-purified using an ATPase-Sepharose column. Addition of a few molecules of the purified antibody per molecule of ATPase was sufficient to inhibit the ATPase activity. Extensively washed ghosts or preincubated pure ATPase sometimes develop an appreciable Mg2+-ATPase activity. In such cases, the antibodies inhibited the Mg2+-ATPase as well as the Ca2+-ATPase. This is consistent with the hypothesis that a portion of the Mg2+-ATPase activity of ghosts is derived from the Ca2+-ATPase. When nitrophenylphosphatase activity was observed, both Mg2+- and Ca2+-stimulated activities were observed. Only the Ca2+ activity was inhibited by the antibodies, confirming that this activity is due to the Ca2+ pump, and suggesting that the Mg2+-nitrophenylphosphatase is due to a separate enzyme. Amounts of antibody comparable to those which inhibited the Ca2+-ATPases had no effect on the Na+-K+-ATPase; 4-fold higher amounts of antibody significantly stimulated the Na+-K+-ATPase, but this effect of the antibody was not specific: Immunoglobulins from the nonimmune serum also significantly stimulated the Na+-K+-ATPase. In resealed erythrocyte membranes, antibodies incorporated into the ghosts inactivated the Ca2+-ATPase, while antibodies added to the outside had no significant effect.
D000251:Adenosine Triphosphatases / Q000097:blood; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D001693:Biological Transport, Active; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium / Q000097:blood; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000276:immunology; D002846:Chromatography, Affinity; D004910:Erythrocyte Membrane / Q000201:enzymology* / Q000276:immunology / Q000648:ultrastructure; D006801:Humans
D000906:Antibodies; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium