Participation of H+ in the Ca2(+)-induced conformational transition of 4-nitro-2,1,3-benzoxadiazole-labeled sarcoplasmic reticulum ATPase.
The binding of Ca2+ to 4-nitro-2,1,3-benzoxadiazole (NBD)-labeled sarcoplasmic reticulum Ca2(+)-ATPase was accelerated markedly when the pH was changed at 11 degrees C from 6.5 to 8.0 at the time of Ca2+ addition. We examined the effect of pH on the enzyme conformational transition by measuring the kinetics of NBD fluorescence rises induced by a pH jump under various ligand conditions. The fast fluorescence rise following a pH jump from 6.0 or 6.5 to various test pHs in the presence and absence of Ca2+ proceeded monoexponentially. The amplitude of this fluorescence rise in the presence of Ca2+ was independent of the test pH, whereas the observed rate constant (kobs) increased markedly as the test pH increased. In contrast, the amplitude of the fast fluorescence rise in the absence of Ca2+ increased with increasing test pH, whereas kobs decreased. MgATP or Mg2+ influenced the pH dependences of these parameters in a complex way except for the amplitudes measured in the presence of Ca2+. These data could be simulated by using a reaction model in which Ca2+ binding is preceded by a rate-limiting enzyme conformational transition from a low to a high NBD fluorescence state and 1 mol each of H+ is liberated before and after this conformational transition. MgATP or Mg2+ appeared to promote this conformational transition by enhancing deprotonation of the enzyme. These results suggest that deprotonation may be the primary event in the activation of the unphosphorylated enzyme by Ca2+.
Wakabayashi
S
S
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Osaka, Japan.
Ogurusu
T
T
Shigekawa
M
M
D009327:4-Chloro-7-nitrobenzofurazan; D000255:Adenosine Triphosphate; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium; D008956:Models, Chemical; D011487:Protein Conformation; D011522:Protons; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D005456:Fluorescent Dyes; D011522:Protons; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D009327:4-Chloro-7-nitrobenzofurazan; D008274:Magnesium; D002118:Calcium