Erythrocyte Ca2+-ATPase: Activation by Enzyme Oligomerization Versus by Calmodulin

Abstract

The subject of our studies is the mechanism of activation of the erythrocyte Ca²⁺-ATPase. Using purified, detergent solubilized enzyme it was found that equivalent maximal Ca²⁺-ATPase activity is obtained either upon addition of calmodulin or upon increase of enzyme concentration. Three independent methods, including Ca20+ -ATPase activity, polarization of the enzyme modified with an external fluorescent probe, and efficiency of fluorescence resonance energy transfer between enzyme molecules have established that the concentration dependent activation is due to enzyme oligomerization. The oligomers bind calmodulin with a lower stoichiometry (0.5 mol calmodulin/mol Ca²⁺-ATPase), higher Ca²⁺ affinity (K_Ca = pCa 7.4), and higher cooperativity for Ca²⁺ (n_H = 2.6) than the monomeric form (stoichiometry =1 mol calmodulin/mol Ca²⁺-ATPase, K_Ca = pCa 7.0, n_H = 1.1). The Ca²⁺ dependence of calmodulin binding and activation of monomers indicates that calmodulin binds before the Ca²⁺-ATPase activity is exhibited, demonstrating that the activation of this enzyme form is totally dependent on calmodulin. In contrast, oligomers reveal very similar Ca²⁺ dependence for calmodulin binding and for Ca²⁺-ATPase activity as well as for Ca²⁺ binding (assessed by tryptophan fluorescence), and for the oligomerization process (assessed by fluorescence energy transfer). The calmodulin antagonist drug 48/80 inhibits the calmodulin dependent activity of the monomers (I₅₀=1.4 μg/ml) but has no effect on the activity of oligomers, confirming that calmodulin plays no role in the activation of the oligomeric enzyme. Our studies indicate that the erythrocyte Ca²⁺-ATPase can be activated by its high affinity, Ca²⁺ dependent binding of calmodulin or by a Ca²⁺ dependent oligomerization process which may involve calmodulin binding site. The red cell Ca²⁺-ATPase has been studied as the most experimentally accessible of the calmodulin regulated plasma membrane Ca²⁺-ATPascs (Schatzmann, 1982; Carafoli and Zurini, 1982). In the red cell the enzyme serves as the only Ca extrusion system to maintain intracellular Ca²⁺ levels near 0.1 µM. Maintenance of this low Ca²⁺ level is important for normal physiological functions. Further, perturbation of intracellular Ca²⁺ has been linked to the etiology of certain disease states, such sickle cell or hypertension (AlJaboree et al., 1984; Penniston, 1984; Vincenzi et al., 1987). The importance of regulation of intracellular Ca²⁺ is reflected in the complexity of regulation of the Ca²⁺-ATPase. In addition to the well established stimulation of the Ca²⁺-ATPase activity by calmodulin (see Schatzmann, 1982) we have recently discovered that the Ca²⁺-ATPase is regulated by enzyme oligomerization (Kosk-Kosicka and Bzdega, 1988). Further, there is evidence for regulation by cAMP dependent phosphorylation (Neyses et al., 1985), proteolysis (Taverna et al., 1980; Enycdi et al., 1987), and acidic phospholipids (Niggli et al., 1981; Choquette et al., 1984). It is the goal of this work to elucidate the mechanism of Ca²⁺-ATPase regulation by calmodulin and by oligomerization, in order to eventually understand the in vivo regulation of the enzyme in health and disease.