title: Effects of pH, Ca2+ and lanthanides on conformation of the sarcoplasmic reticulum Ca(2+)-ATPase catalytic site.
abstract: The conformational changes at the ATP-catalytic site of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase have been studied by the fluorescence of the fluorescein 5-isothiocyanate (FITC) bound to the adenine subsite. The FITC-SR fluorescence parameters have been examined in the pH range 5.7-8.0 in the presence of EGTA, Ca2+ or Ln3+ (La3+, Pr3+, Nd3+, Tb3+, etc.). A quantitative method to calculate the equilibrium between the protein conformers is proposed on the basis of the fluorometric titration curve analysis. The distance Nd(3+)-FITC was estimated to be about 1 nm at pH 6-7 and 1.7 nm at pH 8 which can be interpreted as an increase of the distance between the nucleotide and phosphorylation domains of Ca(2+)-ATPase in alkaline media. These studies suggest that the ligand-stabilized 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 two protein conformations is probably governed by one proton dissociation.
authors.affiliation: Institute of Biological Physics, U.S.S.R. Academy of Sciences, Pushchino, Moscow.
authors.forename: M N
authors.initials: MN
authors.lastname: Ivkova
authors.forename: V V
authors.initials: VV
authors.lastname: Pletnev
authors.forename: M G
authors.initials: MG
authors.lastname: Vinokurov
authors.forename: V A
authors.initials: VA
authors.lastname: Pechatnikov
authors.forename: V G
authors.initials: VG
authors.lastname: Ivkov
authors.forename: I
authors.initials: I
authors.lastname: Jona
authors.forename: J
authors.initials: J
authors.lastname: Fölöp
authors.forename: A
authors.initials: A
authors.lastname: Köver
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000187:drug effects; D016650:Fluorescein-5-isothiocyanate; D006863:Hydrogen-Ion Concentration; D008674:Metals, Rare Earth / Q000494:pharmacology*; D008956:Models, Chemical; D009354:Neodymium / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000494:pharmacology
chemical_list: D008674:Metals, Rare Earth; D013725:Terbium; D009354:Neodymium; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium