Ca2+ binding to sarcoplasmic reticulum ATPase phosphorylated by Pi reveals four thapsigargin-sensitive Ca2+ sites in the presence of ADP.
Sarcoplasmic reticulum (SR) Ca2+-ATPase was phosphorylated by Pi at pH 8.0 in the presence of dimethyl sulfoxide (Me2SO). Under these conditions, it was possible to measure transient 45Ca2+ binding to the phosphoenzyme. Binding reached 1.2 Ca2+ per phosphoenzyme (E-PCax) within 10 min in 30% Me2SO, 20 mM MgCl2 and 0.1 mM Pi and the phosphoenzyme only decreased by 23% during this period. This Ca2+ binding was abolished by thapsigargin, showing that it is associated with functional sites of the Ca2+-ATPase. At 40% Me2SO, simultaneous addition of Ca2+ and ADP increased Ca2+ binding up to almost four Ca2+ per phosphoenzyme (ADPE-PCay), revealing a species bearing simultaneously four Ca2+ sites. Both E-PCax and ADPE-PCay were further identified as distinct species by (2',3'-O-2(2,4,6-trinitrophenyl)adenosine 5'-triphosphate) fluorescence, which revealed long-range modifications in the Ca2+-transport sites induced by ADP binding to E-P. In addition, E-PCax was shown to be a functional intermediate of the cycle leading to ATP synthesis provided that Me2SO was diluted. These findings indicate that more than two functional Ca2+-sites exist on the functional Ca2+-ATPase unit, and that the additional sites become accessible upon ADP addition. This is compatible with a four-site model of the SR Ca2+-ATPase allowing simultaneous binding of Ca2+ at lumenal and cytosolic sites. The stoichiometries for Ca2+ binding found here could either be interpreted as binding of four Ca2+ on a Ca2+-ATPase monomer considered as the functional unit or as binding of two Ca2+ per monomer of a functional dimer.
Vieyra
Adalberto
A
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brasil.
Mintz
Elisabeth
E
Lowe
Jennifer
J
Guillain
Florent
F
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D003198:Computer Simulation; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D009132:Muscles / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012996:Solutions / Q000032:analysis; D013050:Spectrometry, Fluorescence; D019284:Thapsigargin / Q000494:pharmacology*
D002614:Chelating Agents; D010710:Phosphates; D012996:Solutions; D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium; D004121:Dimethyl Sulfoxide