Testing the Versatility of the Sarcoplasmic Reticulum Ca2+-ATPase Reaction Cycle When p-Nitrophenyl Phosphate Is the Substrate
Open Access
- 1 March 2001
- journal article
- Published by Elsevier BV
- Vol. 276 (11), 7998-8004
- https://doi.org/10.1074/jbc.m008648200
Abstract
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.Keywords
This publication has 26 references indexed in Scilit:
- Insight into the Uncoupling Mechanism of Sarcoplasmic Reticulum ATPase Using the Phosphorylating Substrate UTPJournal of Biological Chemistry, 2000
- Reduction in Water Activity Greatly Retards the Phosphoryl Transfer from ATP to Enzyme Protein in the Catalytic Cycle of Sarcoplasmic Reticulum Ca2+-ATPaseJournal of Biological Chemistry, 1996
- The inhibitors thapsigargin and 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone favour the E2 form of the Ca2+, Mg2+‐ATPaseFEBS Letters, 1992
- The vanadate complex of the calcium‐transport ATPase of the sarcoplasmic reticulum, its formation and dissociationEuropean Journal of Biochemistry, 1983
- Effects of p-nitrophenylphosphate on Ca2+ transport in inside-out vesicles from human red-cell membranesBiochimica et Biophysica Acta (BBA) - Biomembranes, 1983
- A model for the reaction pathways of the K+-dependent phosphatase activity of the (Biochimica et Biophysica Acta (BBA) - Biomembranes, 1983
- Vanadate inhibition of the Ca2+‐dependent conformational change of the sarcoplasmic reticulum Ca2+‐ATPaseFEBS Letters, 1982
- Stimulatory and Inhibitory Effects of Dimethyl Sulfoxide and Ethylene Glycol on ATPase Activity and Calcium Transport of Sarcoplasmic MembranesEuropean Journal of Biochemistry, 1977
- Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca2+-pump and Ca2+-binding proteinsBiochimica et Biophysica Acta (BBA) - Biomembranes, 1973
- Regulatory Mechanisms of the Calcium Transport System of Fragmented Rabbit Sarcoplasmic ReticulumThe Journal of general physiology, 1971