Dissecting the hydrolytic activities of sarcoplasmic reticulum ATPase in the presence of acetyl phosphate.
Sarcoplasmic reticulum vesicles and purified Ca(2+)-ATPase hydrolyze acetyl phosphate both in the presence and absence of Ca(2+). The Ca(2+)-independent activity was fully sensitive to vanadate, insensitive to thapsigargin, and proceeded without accumulation of phosphorylated enzyme. Acetyl phosphate hydrolysis in the absence of Ca(2+) was activated by dimethyl sulfoxide. The Ca(2+)-dependent activity was partially sensitive to vanadate, fully sensitive to thapsigargin, and associated with steady phosphoenzyme accumulation. The Ca(2+)/P(i) coupling ratio at neutral pH sustained by 10 mm acetyl phosphate was 0.57. Addition of 30% dimethyl sulfoxide completely blocked Ca(2+) transport and partially inhibited the hydrolysis rate. Uncoupling induced by dimethyl sulfoxide included the accumulation of vanadate-insensitive phosphorylated enzyme. When acetyl phosphate was the substrate, the hydrolytic pathway was dependent on experimental conditions that might or might not allow net Ca(2+) transport. The interdependence of both Ca(2+)-dependent and Ca(2+)-independent hydrolytic activities was demonstrated.
Departamento de Bioquimica y Biologia Molecular A, Edificio de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30071 Murcia, Spain.
Fernando
F
Soler
Maria-Isabel
MI
Fortea
Antonio
A
Lax
Francisco
F
Fernández-Belda
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D022162:Cytoplasmic Vesicles / Q000737:chemistry / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D018482:Muscle, Skeletal / Q000166:cytology; D010755:Organophosphates / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D012997:Solvents; D019284:Thapsigargin / Q000378:metabolism; D014638:Vanadates / Q000378:metabolism
D002132:Calcium Radioisotopes; D004791:Enzyme Inhibitors; D010755:Organophosphates; D012997:Solvents; D014638:Vanadates; C011632:acetyl phosphate; D019284:Thapsigargin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium; D004121:Dimethyl Sulfoxide