Testing the versatility of the sarcoplasmic reticulum Ca2+-ATPase reaction cycle when p-nitrophenyl phosphate is the substrate

A detailed characterization of p-nitrophenyl phosphate as energy-donor Substrate for the sarcoplasmic reticulum Ca2+-ATPase was undertaken in this study. The fact that p-nitrophenyl phosphate can be hydrolyzed in the presence or absence of Ca2+ by the purified enzyme is consistent with the observed phenomenon of intramolecular uncoupling. Under the most favorable conditions, which include neutral pH, intact microsomal vesicles, and low free Ca2+ in the lumen, the Ca2+/P-i coupling ratio was 0.6. A rise or decrease in pH, high free Ca2+ in the lumenal space, or the addition of dimethyl sulfoxide increase the intramolecular uncoupling. Alkaline pH and/or high free Ca2+ in the lumen potentiate the accumulation of enzyme conformations with high Ca2+ affinity, Acidic pH and/or dimethyl sulfoxide favor the accumulation of enzyme conformations with low Ca2+ affinity. Under standard assay conditions, two uncoupled routes, together with a coupled route, are operative during the hydrolysis of p-nitrophenyl phosphate in the presence of Ca2+. The prevalence of any one of the uncoupled catalytic cycles is dependent on the working conditions. The proposed reaction scheme constitutes a general model for understanding the mechanism of intramolecular energy uncoupling.