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J. Biol. Chem., Vol. 276, Issue 11, 7998-8004, March 16, 2001

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Testing the Versatility of the Sarcoplasmic Reticulum Ca²⁺-ATPase Reaction Cycle When p-Nitrophenyl Phosphate Is the Substrate^*

Francisco Fernandez-Belda, Maria-Isabel Fortea, and Fernando Soler

From the Departamento de Bioquimica y Biologia Molecular A, Edificio de Veterinaria, Universidad de Murcia en Espinardo, 30071 Murcia, Spain

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.

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