Modificações estruturais da Ca²⁺ATPase de eritrócito em diferentes estados formacionais estudadas por espectroscopia de fluorescência

Abstract

The structure of the plasma membrane erythrocyte Ca²-ATPase was studied using fluorescence techniques. Fluorescence spectroscopy was combined with hydrostatic pressure to allow the study of the dissociation of the oligomeric ATPase. The erythrocyte Ca²-ATPase was shown to be a dimer with a dissociation constant (KD) between 6-9 x 10-6 M. The volume change upon dissociation was found to be approximately - 50 ml/mol. This relatively small volume change may indicate that hydrophobic interactions predominate over electrostatic interactions in the interface between the subunits. The stabilization of either the E1 ATPase conformation (by the addition of Ca²⁺) or E2 (by the addition of vanadate) favoured, respectively, the associated or the dissociated States. Calmodulin did not promote any additional effect on the dissociation over the effect promoted by Ca²⁺. The results concerning the effects of ligands on the oligomerization of the erythrocyte ATPase suggest that calmodulin does not alter the equilibrium between the two major conformational States E1 and E2. Fluorescence lifetime measurements showed that calmodulin stabilizes the enzyme in a different conformation as compared to the conformation stabilized by Ca²⁺ alone. The study of the effects of organic osmolytes on the structure and function of the erythrocyte Ca²⁺-ATPase indicated that betaine - a representative methylamine - was able to protect against the uncoupling between Ca²⁺ transport and ATP hydrolysis induced by urea. The conformational changes promoted by betaine appeared to derive from direct interactions between betaine and the ATPase. In the presence of betaine an increase in compactation of the more hydrophilic domains of the ATPase was observed. On the other hand, betaine decreased the compactation at hydrophobic internal domains of the protein, which may include the amino acids that are in contact with the phospholipids of the native membrane.