Calmodulin affinity chromatography yields a functional purified erythrocyte (Ca2+ + Mg2+)-dependent adenosine triphosphatase

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Abstract
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 MW 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.degree. C and in the presence of calmodulin the purified ATPase had a specific activity of 10.1 .mu.mol/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-specificity of the ATPase, since the lipids can easily be exchanged without a significant loss of activity. A purification procedure described by Niggli, Penniston and Carafoli resulted in an enzyme that, indeed, was pure, but was lacking modulation by calmodulin.