Fluorescence studies on calmodulin binding to erythrocyte calcium ATPase in different oligomerization states

Abstract

The fluorescent spinach calmodulin derivative 2-(4-maleimidoanilino)napthalene-6-sulfonic acid-calmodulin (MIANS-CaM) was used to investigate calmodulin interaction with the purified, detergent-solubilized erythrocyte Ca2+-ATPase. Previous studies have shown that the Ca2+-ATPase exists in equilibria between monomeric and oligomeric forms. We report here that MIANS-CaM binds to both enzyme forms in a Ca2+-dependent manner, with a .apprx. 50% fluorescence enhancement. These findings confirm our previous observation that enzyme oligomers retain their ability to bind calmodulin, even though they are fully activated in the absence of calmodulin. The Ca2+ dependence of MIANS-CaM binding to monomeric Ca2+-ATPase is of higher affinity (K1/2 = 0.09 .mu.M Ca2+) and less cooperative (nH = 1.1) than the Ca2+ dependence of enzyme activation by MIANS-CaM (K1/2 = 0.26 .mu.M Ca2+, nH = 2.8). These Ca2+ dependences and the order of events, in which calmodulin binding precedes enzyme activation, demonstrate that calmodulin indeed could be a physiological activator of the monomeric enzyme. The calmodulin dependence of calmodulin binding to oligomeric Ca2+-ATPase occurs at even lower levels of Ca2+ (K1/2 = 0.04 .mu.M Ca2+), in a highly cooperative fashion (nH = 2.3), and essentially in parallel with enzyme activation (K1/2 = 0.05 .mu.M Ca2+, nH = 2.9). The observed differences between monomers and oligomers suggest that the oligomerized Ca2+-ATPase is in a conformation necessary for efficient, cooperative calcium binding at nanomolar Ca2+ which the monomeric enzyme acquires only upon interaction with calmodulin. Fluorescence titrations of Ca2+-ATPase oligomers and monomers with MIANS-CaM reveal significant difference, indicating that the oligomer is saturated near 0.5 mol of calmodulin/mol of Ca2+-ATPase while the monomer is saturated near 1 mol of calmodulin/mol of Ca2+-ATPase. These results suggest the possibility that oligomerization of the Ca2+-ATPase monomers results in the elimination of one calmodulin binding site per every two associated enzyme molecules.