Testing the versatility of the sarcoplasmic reticulum Ca(2+)-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 Ca(2+)-ATPase was undertaken in this study. The fact that p-nitrophenyl phosphate can be hydrolyzed in the presence or absence of Ca(2+) 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 Ca(2+) in the lumen, the Ca(2+)/P(i) coupling ratio was 0.6. A rise or decrease in pH, high free Ca(2+) in the lumenal space, or the addition of dimethyl sulfoxide increase the intramolecular uncoupling. Alkaline pH and/or high free Ca(2+) in the lumen potentiate the accumulation of enzyme conformations with high Ca(2+) affinity. Acidic pH and/or dimethyl sulfoxide favor the accumulation of enzyme conformations with low Ca(2+) 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 Ca(2+). 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.
Fernandez-Belda
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F
Departamento de Bioquimica y Biologia Molecular A, Edificio de Veterinaria, Universidad de Murcia en Espinardo, 30071 Murcia, Spain. fbelda@um.es
Fortea
M I
MI
Soler
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F
D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009596:Nitrophenols; D063065:Organophosphonates / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D019284:Thapsigargin / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D009596:Nitrophenols; D063065:Organophosphonates; C085778:4-nitrophenyl phosphonate; D014638:Vanadates; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium; D004121:Dimethyl Sulfoxide