Association between human erythrocyte calmodulin and the cytoplasmic surface of human erythrocyte membranes

This report describes Ca²⁺-dependent binding of ¹²⁵I-labeled calmodulin (¹²⁵I-CaM) to erythrocyte membranes and indentification of two new CaM-binding proteins. Erythrocyte CaM labeled with ¹²⁵I-Bolton Hunter reagent fully activated erythrocyte (Ca²⁺ + Mg²⁺)-ATPase. ¹²⁵I-CaM bound to CaM depleted membranes in a Ca²⁺-dependent manner with a K(a) of 6 x 10^-8 M Ca²⁺ and maximum binding at 4 x 10^-7 M Ca²⁺. Only the cytoplasmic surface of the membrane bound ¹²⁵I-CaM. Binding was inhibited by unlabeled CaM and by trifluoperazine. Reduction of the free Ca²⁺ concentration or addition of trifluoperazine caused a slow reversal of binding. Nanomolar ¹²⁵I-CaM required several hours to reach binding equilibrium, but the rate was much faster at higher concentrations. Scatchard plots of binding were curvilinear, and a class of high affinity sites was identified with a K(D) of 0.5 nM and estimated capacity of 400 sites per cell equivalent for inside-out vesicles (IOVs). The high affinity sites of IOVs most likely correspond to Ca²⁺ transporter since: (a) K(a) of activation of (Ca²⁺ + Mg²⁺)-ATPase and K(D) for binding were nearly identical, and (b) partial digestion of IOVs with α-chymotrypsin produced activation of the (Ca²⁺ + Mg²⁺)-ATPase with loss of the high affinity sites. ¹²⁵I-CaM bound in solution to a class of binding proteins (K(D) ~ 55 nM, 7.3 pmol per mg of ghost protein) which were extracted from ghosts by low ionic strength incubation. Soluble binding proteins were covalently cross-linked to ¹²⁵I-CaM with Lomant's reagent, and 2 bands of 8,000 and 40,000 M(r)(M(r) of CaM subtracted) and spectrin dimer were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. The 8,000 and 40,000 M(r) proteins represent a previously unrecognized class of CaM-binding sites which may mediate unexplained Ca²⁺-induced effects in the erythrocyte.