CONFORMATIONAL-CHANGES AT THE ATP-CATALYT IC SITE OF THE RECONSTITUTED SARCOPLASMIC-RETICULUM CA-ATPASE UNDER THE ACTION OF PH, CA2+ AND LANTANIDES

Conformational changes at the ATP-catalytic site of the sarcoplasmic r eticulum Ca-ATPase reconstituted in proteoliposomes have been studied by the fluorescence of the fluorescein 5-isothiocyanate (FITC). It bin ds to Lys-515 at the adenine binding site of the nucleotide domain. Th e FITC-Ca-ATPase fluorescence parameters have been examined in the pH range 5,7-8,0 in the presence of EGTA, Ca2+, lantanides. The quantitat ive method was used to calculate the equilibrium between the protein c onformers E-1 and E-2. It is based on the analysis of fluorometric tit ration curves. Lantanides were estimate the distances between nucleoti de and phosphorylation domains in the pH range 5,7-8,0. The distance N d3+-FITC was estimated to be about 1 nm at pH 6 and 1,7 nm at pH 8, wh ich can be interpreted as an increase in the distance between the nucl eotide and phosphorylation domains of Ca-ATPase in alkaline media. The se studies suggest that the ligand stabilized by the E-1-form of Ca2+- ATPase can exist in two conformational states with the closed and open ed interdomain cleft: in the pH range 5,7-8,0. The pH-dependence of th e ratio of these states correlates with that of the E-1<----> E-2 equi librium without ligands. These dependences were approximated by simple Kenderson-Hasselbach equations with pK 7,0+/-0,1,i.e. the transition between the two protein confer mations is probably governed by one pro ton dissociation. Model experiments were used to determine the lantani de binding with proteoliposome lipid part. The Nd3+ association consta nt at the substrate site has been estimated to be 1,5 10(5)M(-1) at pH 6,0; 1,0.10(5) M-1 at pH 7,0 and 0,7.10(5) M-1 at pH 8,0.