Functional effect of hydrogen peroxide on the sarcoplasmic reticulum membrane: uncoupling and irreversible inhibition of the Ca2+-ATPase protein.
The chemical treatment of sarcoplasmic reticulum vesicles with H2O2 affects both Ca2+ transport and the hydrolytic activity supported by the Ca2+-ATPase protein. Ca2+ transport was much more sensitive to inhibition than ATPase activity and the decrease in Ca2+ transport was not the result of an increase in membrane permeability. The Ca2+/Pi uncoupling can be attributed to the own catalytic mechanism of the enzyme. Under conditions of high uncoupling, Ca2+ binding to the transport sites was barely affected and accumulation of phosphorylated species during the enzyme cycling gave almost maximal levels. These are features defining intramolecular uncoupling mediated by a phosphorylated form of the enzyme. Severe inhibition of the hydrolytic activity was observed when higher peroxide concentrations and leaky vesicles were used. These experimental conditions diminished maximal Ca2+ binding and the steady-state phosphoenzyme level. The low hydrolytic activity can be ascribed to a decrease in the rate of enzyme dephosphorylation.
Sánchez
Sonia
S
Departamento de Bioquímica y Biología Molecular A, Facultad de Veterinaria, Universidad de Murcia en Espinardo, 30071 Espinardo, Murcia, Spain.
Fernández-Belda
Francisco
F
Soler
Fernando
F
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors*; D002374:Catalase / Q000494:pharmacology; D002384:Catalysis; D004533:Egtazic Acid / Q000494:pharmacology; D005260:Female; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018656:Muscle Fibers, Fast-Twitch / Q000201:enzymology; D018482:Muscle, Skeletal / Q000201:enzymology; D010766:Phosphorylation; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects* / Q000201:enzymology*
D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide; D002374:Catalase; D000252:Calcium-Transporting ATPases; D002118:Calcium