Insight into the uncoupling mechanism of sarcoplasmic reticulum ATPase using the phosphorylating substrate UTP.
Ca(2+) transport and UTP hydrolysis catalyzed by sarcoplasmic reticulum Ca(2+)-ATPase from skeletal muscle was studied. A passive Ca(2+) load inside microsomal vesicles clearly decreased the net uptake rate and the final accumulation of Ca(2+) but not the UTP hydrolysis rate, causing energy uncoupling. In the absence of passive leak, the Ca(2+)/P(i) coupling ratio was 0.7-0.8. UTP hydrolysis did not maintain a rapid component of Ca(2+) exchange between the cytoplasmic and lumenal compartments as occurs with ATP. The uncoupling process in the presence of UTP is associated with: (i) the absence of a steady state accumulation of ADP-insensitive phosphoenzyme; (ii) the cytoplasmic dissociation of Ca(2+) bound to the ADP-sensitive phosphoenzyme; and (iii) the absence of enzyme inhibition by cyclopiazonic acid. All these characteristics confirm the lack of enzyme conformations with low Ca(2+) affinity and point to the existence of an uncoupling mechanism mediated by a phosphorylated form of the enzyme. Suboptimal coupling values can be explained in molecular terms by the proposed functional model.
Fortea
M I
MI
Departamento de Bioquimica y Biologia Molecular A, Edificio de Veterinaria, Universidad de Murcia en Espinardo, 30071 Murcia, Spain.
Soler
F
F
Fernandez-Belda
F
F
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D005260:Female; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000201:enzymology; D008956:Models, Chemical; D018482:Muscle, Skeletal / Q000201:enzymology; D010070:Oxalates / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013997:Time Factors; D014475:Uncoupling Agents / Q000378:metabolism; D014544:Uridine Triphosphate / Q000378:metabolism*
D010070:Oxalates; D014475:Uncoupling Agents; D000251:Adenosine Triphosphatases; D002118:Calcium; D014544:Uridine Triphosphate