A kinetic study of the interaction of vanadate with the Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum.
Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum was inhibited by preincubation with vanadate. When the inhibited enzyme was preincubated in the presence of vanadate and assayed in its absence, a slow reactivation process was observed. This slow, hysteretic, process was exploited to study th...
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pubmed-11440222005-09-07 A kinetic study of the interaction of vanadate with the Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum. Ortiz, A García-Carmona, F García-Cánovas, F Gómez-Fernández, J C Biochem J Research Article Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum was inhibited by preincubation with vanadate. When the inhibited enzyme was preincubated in the presence of vanadate and assayed in its absence, a slow reactivation process was observed. This slow, hysteretic, process was exploited to study the influence of Ca2+ and ATP on the dissociation of vanadate. Ca2+ alone slowly displaced vanadate from the inhibited enzyme, and a rate constant of 0.1 min-1, at 25 degrees C, was calculated for this re-activation process. However, ATP re-activated with an apparent constant that hyperbolically depended on ATP concentration, and from it a rate constant for vanadate dissociation induced by ATP of 0.5 min-1 was calculated. It is deduced from the kinetic studies that ATP binds to the enzyme-vanadate complex, forming a ternary complex, with a dissociation constant of 4 microM, and that this binding accelerates vanadate dissociation. Binding experiments with [14C]ATP showed that ATP binds to the enzyme-vanadate complex with a dissociation constant of 12 microM, i.e. the affinities calculated with the isotope technique and the kinetic procedure are of the same order of magnitude. 1984-07-01 /pmc/articles/PMC1144022/ /pubmed/6147134 Text en |
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Research Article Ortiz, A García-Carmona, F García-Cánovas, F Gómez-Fernández, J C A kinetic study of the interaction of vanadate with the Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum. |
description |
Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum was inhibited by preincubation with vanadate. When the inhibited enzyme was preincubated in the presence of vanadate and assayed in its absence, a slow reactivation process was observed. This slow, hysteretic, process was exploited to study the influence of Ca2+ and ATP on the dissociation of vanadate. Ca2+ alone slowly displaced vanadate from the inhibited enzyme, and a rate constant of 0.1 min-1, at 25 degrees C, was calculated for this re-activation process. However, ATP re-activated with an apparent constant that hyperbolically depended on ATP concentration, and from it a rate constant for vanadate dissociation induced by ATP of 0.5 min-1 was calculated. It is deduced from the kinetic studies that ATP binds to the enzyme-vanadate complex, forming a ternary complex, with a dissociation constant of 4 microM, and that this binding accelerates vanadate dissociation. Binding experiments with [14C]ATP showed that ATP binds to the enzyme-vanadate complex with a dissociation constant of 12 microM, i.e. the affinities calculated with the isotope technique and the kinetic procedure are of the same order of magnitude. |
author |
Ortiz, A García-Carmona, F García-Cánovas, F Gómez-Fernández, J C |
author_facet |
Ortiz, A García-Carmona, F García-Cánovas, F Gómez-Fernández, J C |
author_sort |
Ortiz, A |
title |
A kinetic study of the interaction of vanadate with the Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum. |
title_short |
A kinetic study of the interaction of vanadate with the Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum. |
title_full |
A kinetic study of the interaction of vanadate with the Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum. |
title_fullStr |
A kinetic study of the interaction of vanadate with the Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum. |
title_full_unstemmed |
A kinetic study of the interaction of vanadate with the Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum. |
title_sort |
kinetic study of the interaction of vanadate with the ca2+ + mg2+-dependent atpase from sarcoplasmic reticulum. |
publishDate |
1984 |
url |
https://ncbi.nlm.nih.gov/pmc/articles/PMC1144022/ https://ncbi.nlm.nih.gov/pubmed/6147134 |
_version_ |
1744687478143451136 |