Calmodulin affinity chromatography yields a functional purified erythrocyte (Ca+ + Mg2+)-dependent adenosine triphosphatase.
The (Ca2+ + Mg2+)-dependent ATPase of human erythrocyte membranes was solubilized with deoxycholate and purified by calmodulin affinity chromatography to yield a functional enzyme. The method gave an enzyme purified 207-fold as compared with that of the erythrocyte membranes. The molecular weight of the ATPase was in the range 135 000-150 000, as revealed by a single major band after electrophoresis on dodecyl sulphate/polyacrylamide gels. The isolated enzyme was highly sensitive to calmodulin, since the activity was increased about 9-fold. At 37 degrees C and in the presence of calmodulin the purified ATPase had a specific activity of 10.1 mumol/min per mg of protein. Triton X-100 or deoxycholate stimulated the calmodulin-deficient enzyme in a concentration-dependent fashion whereby the calmodulin-sensitivity was lost. The purification method is suitable for studying the lipid-sensitivity of the ATPase, since the lipids can easily be exchanged without a significant loss of activity. A purification procedure described by Niggli, Penniston & Carafoli [(1979) J. Biol. Chem. 254, 9955-9958] resulted in an enzyme that indeed was pure but was lacking a predominant feature, namely the modulation by calmodulin.
D000252:Calcium-Transporting ATPases / Q000097:blood / Q000302:isolation & purification*; D002147:Calmodulin / Q000494:pharmacology; D002846:Chromatography, Affinity; D003902:Detergents; D004789:Enzyme Activation / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D008055:Lipids; D008274:Magnesium
D002147:Calmodulin; D003902:Detergents; D008055:Lipids; D000252:Calcium-Transporting ATPases; D008274:Magnesium