1,2-Dichlorobenzene affects the formation of the phosphoenzyme stage during the catalytic cycle of the Ca2+-ATPase from sarcoplasmic reticulum

Abstract

Background: 1,2-Dichlorobenzene (1,2-DCB) is a benzene-derived molecule with two Cl atoms that is commonly utilized in the synthesis of pesticides. 1,2-DCB can be absorbed by living creatures and its effects on naturally-occurring enzymatic systems, including the effects on Ca2+-ATPases, have been poorly studied. Therefore, we aimed to study the effect of 1,2-DCB on the Ca2+-ATPase from sarcoplasmic reticulum (SERCA), a critical regulator of intracellular Ca2+ concentration. Results: Concentrations of 0.05-0.2 mM of 1,2-DCB were able to stimulate the hydrolytic activity of SERCA in a medium-containing Ca2+-ionophore. At higher concentrations (0.25-0.75 mM), 1,2-DCB inhibited the ATP hydrolysis to ~80 %. Moreover, ATP hydrolysis and Ca2+ uptake in a medium supported by K-oxalate showed that starting at 0.05 mM,1,2-DCB was able to uncouple the ratio of hydrolysis/Ca2+ transported. The effect of this compound on the integrity of the SR membrane loaded with Ca2+ remained unaffected. Finally, the analysis of phosphorylation of SERCA by [γ-32P]ATP, starting under different conditions at 0° or 25 °C showed a reduction in the phosphoenzyme levels by 1,2-DCB, mostly at 0 °C. Conclusions: The temperature-dependent decreased levels of phosphoenzyme by 1,2-DCB could be due to the acceleration of the dephosphorylation mechanism - E2P · Ca2 state to E2 and Pi, which explains the uncoupling of the ATP hydrolysis from the Ca2+ transport.