[Conformational changes at the ATP-catalytic site of the reconstituted sarcoplasmic reticulum Ca-ATPase under th action of pH, Ca2+, and lanthanides].
Conformational changes at the ATP-catalytic site of the sarcoplasmic reticulum Ca-ATPase reconstituted in proteoliposomes have been studied by the fluorescence of the fluorescein 5-isothiocyanate (FITC). It binds to Lys-515 at the adenine binding site of the nucleotide domain. The FITC-Ca-ATPase fluorescence parameters have been examined in the pH range 5,7-8,0 in the presence of EGTA, Ca2+, lantanides. The quantitative method was used to calculate the equilibrium between the protein conformers E1 and E2. It is based on the analysis of fluorometric titration curves. Lantanides were used to estimate the distances between nucleotide and phosphorylation domains in the pH range 5.7-8.0. The distance between Nd(3+)-FITC was estimated to be about 1 nm at pH 6 and 1.7 nm at pH 8, which can be interpreted as an increase in the distance between the nucleotide and phosphorylation domains of Ca-ATPase in alkaline media. These studies suggest that the ligand stabilized by the E1-form of Ca(2+)-ATPase can exist in two conformational states with the closed and opened interdomain cleft in the pH range 5.7-8.0. The pH-dependence of the ratio of these states correlates with that of the E1<-->E2 equilibrium without ligands. These dependences were approximated by simple Henderson-Hasselbach equations with pK 7.0 +/- 0.1, i.e. the transition between the two protein conformations is probably governed by one proton dissociation. Model experiments were used to determine the lantanide binding with proteoliposome lipid part. The Nd3+ association constant 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.
Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
M G
MG
Vinokurov
M N
MN
Ivkova
V A
VA
Pechatnikov
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D008674:Metals, Rare Earth / Q000494:pharmacology*; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D008674:Metals, Rare Earth; D000252:Calcium-Transporting ATPases; D002118:Calcium