Ca2+ binding to sarcoplasmic reticulum ATPase phosphorylated by P-i reveals four thapsigargin-sensitive Ca2+ sites in the presence of ADP

摘要

Sarcoplasmic reticulum (SR) Ca2+-ATPase was phosphorylated by P-i at pH 8.0 in the presence of dimethyl sulfoxide (Me2SO). Under these conditions, it was possible to measure transient Ca-45(2+) 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 P-i 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. (C) 2004 Elsevier B.V. All rights reserved.