Assaying P-Type ATPases Reconstituted in Liposomes.

  title={Assaying P-Type ATPases Reconstituted in Liposomes.},
  author={H. Apell and Bojana Damnjanovi{\'c}},
  journal={Methods in molecular biology},
Reconstitution of P-type ATPases in unilamellar liposomes is a useful technique to study functional properties of these active ion transporters. Experiments with such liposomes provide an easy access to substrate-binding affinities of the ion pumps as well as to the lipid and temperature dependence of the pump current. Here, we describe two reconstitution methods by dialysis and the use of potential-sensitive fluorescence dyes to study transport properties of two P-type ATPases, the Na,K-ATPase… Expand
Mechanistic principles of ion transport in the Na,K-ATPase
  • H. Apell
  • Chemistry
  • Russian Journal of Electrochemistry
  • 2017
The Na,K-ATPase is a member of the P-type ATPase family and a primary active ion transporter for Na+ and K+ ions in the cytoplasmic membrane of virtually all animal cells. Considerable progress inExpand
The KdpFABC complex – K+ transport against all odds
Evidence supporting contradictory models of the KdpFABC complex is discussed and key experiments are identified needed to resolve discrepancies and produce a unified model for this fascinating mechanistic hybrid. Expand


The vanadate-sensitive ATPase of Streptococcus faecalis pumps potassium in a reconstituted system.
The vanadate-sensitive ATPase of Streptococcus faecalis, purified to homogeneity, was reconstituted into soybean phospholipid vesicles in a functional state and appears to function as a potential regulated, ATP-driven pump that serves in electrogenic potassium accumulation by the bacterial cell. Expand
Electrogenic K+ transport by the Kdp-ATPase of Escherichia coli.
The experiments demonstrate the electrogeneity of the Kdp-ATPase in a purified reconstituted system and measured electrical signals as well as the dye measurements correspond to the transport of positive charge to the intracellular face of the protein. Expand
Preparation of Na,K-ATPase-containing liposomes with predictable transport properties by a procedure relating the Na,K-transport capacity to the ATPase activity.
A procedure is developed which allows to prepare standardized Na,K-ATPase-liposomes with predictable transport properties and the transport properties are related to the Na, K- ATPase activity preset before dialysis. Expand
Mechanistic analysis of the pump cycle of the KdpFABC P-type ATPase.
Various mechanistic pump cycle models were derived from the general Post-Albers scheme of P-type ATPases and discussed in the framework of the experimental evidence to propose a possible molecular pump cycle for KdpFABC. Expand
KdpFABC reconstituted in Escherichia coli lipid vesicles: substrate dependence of the transport rate.
The results indicate that high cytoplasmic K(+) concentrations have an inhibitory effect on the KdpFABC complex, and ion pumping was activated by addition of ATP to the external medium which corresponds to the cy toplasm under physiological conditions. Expand
Quantitative analysis of pump-mediated fluxes in reconstituted lipid vesicles
Abstract Reconstituted vesicles with built-in ion pumps (or other transport proteins) are usually heterogeneous with respect to size and with respect to the number of pump molecules present in theExpand
Optical study of active ion transport in lipid vesicles containing reconstituted Na,K-ATPase
The optical method has a higher time resolution than tracer-flux experiments and allows an accurate determination of initial flux rates and the amplitude of the fast signal change increases with decreasing passive ion permeability of the vesicle membrane. Expand
Na,K-ATPase in artificial lipid vesicles. Comparison of Na,K and Na-only pumping mode.
Strong evidence is found that the Na,K-ATPase from rabbit kidney outer medulla exhibits a stoichiometry of 3Na+cyt/2Na+ext/1ATP, i.e. the extracellular (= intravesicular) Na+ has a potassium-like effect. Expand
[26] Isolation of (Na+ + K+)-ATPase
Publisher Summary the discovery of (Na + + K + )-ATPase, intensive studies have been carried out to elucidate its rote in the active transport of sodium and potassium. The enzyme has not beenExpand
Surface Charges of the Membrane Crucially Affect Regulation of Na,K-ATPase by Phospholemman (FXYD1)
The positively charged C-terminal helix of FXYD1 appears to be highly mobile and may interact with the cytoplasmic N domain of the α-subunit, the interaction being strongly affected by phosphorylation at Ser68 and the surface charge of the membrane. Expand