Year-end Sale % OFF 
Abstract
We used partially purified Na+/K+-ATPase from pig kidney to study dephosphorylation, occlusion, and ATPase activity in the same enzyme preparation and in media of identical composition containing 10 microM ATP and different concentrations of Rb+, used as a K+ congener. The experiments were performed using a rapid-mixing apparatus with a time resolution of 3.5 ms. The main findings were as follows. (i) At sufficiently low Rb+ concentration the initial rate of dephosphorylation was higher than that of occlusion, (ii) as [Rb+] tended to zero the slope of the time course of occlusion but not that of the time course of dephosphorylation approached zero and, (iii) as Rb+ concentration increased, ATPase activity first increased and, after passing through a maximum, tended to a value that was lower than that observed in media without Rb+. None of these results is compatible with the currently held idea that binding of a single Rb+ to the E2P conformer of the ATPase does not modify the rate of dephosphorylation and strongly suggest that a single Rb+ does promote dephosphorylation through a mechanism that is not stoichiometrically coupled to Rb+ occlusion. If this mechanism is included in the currently accepted scheme for ATP hydrolysis by the Na+/K+-ATPase, a reasonable prediction of the experimental results is obtained.
Highlights
To zero the release of Kϩ becomes the slowest step of the Naϩ/KϩATPase cycle
ATPase couples the hydrolysis of ATP to the transport of three intracellular sodium ions in exchange for two extracellular potassium ions
The initial rates of Rbϩ-dependent dephosphorylation and of Rbϩ occlusion were calculated from (i) the solution at t ϭ 0 of the first derivative of the functions fitted to the complete time courses (Fig. 2), (ii) the slope of the straight lines fitted to data of occluded Rbϩ and of phosphoenzyme concentrations measured at the time of addition of Rbϩ (t ϭ 0) and after a 93-ms-long incubation
Summary
The experiments were started adding the desired concentrations of Rbϩ to the incubation medium in which the enzyme (final concentration 40 –50 g protein/ ml) performed steady-state Naϩ-ATPase activity. Because acid denaturation by chemical quenching releases occluded Rbϩ, occlusion was measured after stopping the reaction by rapid cooling-and-washing following the procedure developed in our laboratory [9]. The initial rates of Rbϩ-dependent dephosphorylation and of Rbϩ occlusion were calculated from (i) the solution at t ϭ 0 of the first derivative of the functions fitted to the complete time courses (Fig. 2), (ii) the slope of the straight lines fitted to data of occluded Rbϩ and of phosphoenzyme concentrations measured at the time of addition of Rbϩ (t ϭ 0) and after a 93-ms-long incubation.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
