Functional effect of hydrogen peroxide on the sarcoplasmic reticulum membrane: uncoupling and irreversible inhibition of the Ca 2+-ATPase protein

Abstract

The chemical treatment of sarcoplasmic reticulum vesicles with H 2O 2 affects both Ca 2+ transport and the hydrolytic activity supported by the Ca 2+-ATPase protein. Ca 2+ transport was much more sensitive to inhibition than ATPase activity and the decrease in Ca 2+ transport was not the result of an increase in membrane permeability. The Ca 2+/P i uncoupling can be attributed to the own catalytic mechanism of the enzyme. Under conditions of high uncoupling, Ca 2+ 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 Ca 2+ binding and the steady-state phosphoenzyme level. The low hydrolytic activity can be ascribed to a decrease in the rate of enzyme dephosphorylation.