Effects of pH, Ca2+ and lanthanides on conformation of the sarcoplasmic reticulum Ca(2+)-ATPase catalytic site.
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.
Ivkova
M N
MN
Institute of Biological Physics, U.S.S.R. Academy of Sciences, Pushchino, Moscow.
Pletnev
V V
VV
Vinokurov
M G
MG
Pechatnikov
V A
VA
Ivkov
V G
VG
Jona
I
I
Fölöp
J
J
Köver
A
A
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
D008674:Metals, Rare Earth; D013725:Terbium; D009354:Neodymium; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium