We compared ATP-induced K+ translocation in WT Kdp and a transport deficient mutant and determined the EC50 for ATP.
Background and Assay Description:
We performed a single solution exchange workflow by applying ATP in presence of 300 mM KCl to measure ATP-driven K+ translocation in KdpFABC. By applying different ATP concentrations in the range between 300 μM and 3 µM we could determine the EC50. The same assay was performed using D307A Kdp, a mutant which does not catalyze K+ translocation. Here, no current signal was observed for all ATP concentrations (red traces).
Author: Nanion Technologies in collaboration with David Stokes, NYU School of Medicine
Target: KdpFABC complex
Family/Type: KdpB: p-type ATPase; KdpA: channel-like, SKT superfamily
Mode of transport: ATP-driven K+ pump
Organism: Escherichia coli
Sample: Proteoliposomes from purified protein (E.coli Polar Lipid Extract : POPC 3:1), LPR 10
Expression: Recombinant expression in Escherichia coli
Platform: SURFE²R N1