Mechanism for activation of the 4-nitrobenzo-2-oxa-1,3-diazole-labeled sarcoplasmic reticulum ATPase by Ca2+ and its modulation by nucleotides.
The mechanism for activation of sarcoplasmic reticulum ATPase by Ca2+ was investigated in 2 mM MgCl2 and 0.1 M KCl at pH 6.5 and 11 degrees C by using enzyme preparations in which a specific amino acid residue (Cys-344) was labeled with 4-nitrobenzo-2-oxa-1,3-diazole (NBD) [Wakabayashi, S., Imagawa, T., & Shigekawa, M. (1990) J. Biochem. (Tokyo) 107, 563-571]. We compared the kinetics of binding and dissociation of Ca2+ from the enzyme with those of the accompanying NBD fluorescence changes. The fluorescence rise following addition of Ca2+ proceeded monoexponentially. At 2-100 microM Ca2+ and in the absence of nucleotides, the Ca2(+)-induced fluorescence rise and Ca2+ binding to the enzyme proceeded at similar rates, which were almost independent of the Ca2+ concentration. In contrast, the fluorescence decrease induced by Ca2+ removal was slower than the Ca2+ dissociation, and both of these processes were inhibited markedly by increasing medium Ca2+. ATP by binding at 1 mol/mol of the phosphorylation site markedly accelerated both the Ca2(+)-induced fluorescence rise and Ca2+ binding, ADP and AMPPNP but not GTP also being effective. In contrast, ADP minimally affected the NBD fluorescence decrease and the Ca2+ dissociation. These data are consistent with a reaction model in which binding of Ca2+ occurs after the conformational transition of the free enzyme from a state (E2) having low affinity for Ca2+ to one (E1) having high affinity for Ca2+ and in which ATP bound at the catalytic site of E2, whose affinity for ATP is about 30-fold less than that of E1, accelerates this conformational transition.
Wakabayashi
S
S
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Osaka, Japan.
Shigekawa
M
M
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005456:Fluorescent Dyes; D007700:Kinetics; D009711:Nucleotides / Q000494:pharmacology*; D010069:Oxadiazoles; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D009711:Nucleotides; D010069:Oxadiazoles; C011514:4-nitrobenzofurazan; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D002118:Calcium