Characterization of (Na+ + K+)-ATPase-liposomes. III. Controlled activation and inhibition of symmetric pumps by timed asymmetric ATP, RbCl, and cardiac glycoside addition

Rey, H. G.; Moosmayer, M.; Anner, Béatrice M.

doi:10.1016/0005-2736(87)90274-4

Cited by 10 publications

(4 citation statements)

References 30 publications

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“…The dialysis procedure yields unilamellar vesicles with a diameter of about 90 nm in which the enzyme is incorporated in random orientation (Apell et al, 1985;Rey, Moosmayer & Anner, 1987). Addition of caged ATP (which may be assumed to be virtually membrane-impermeable) to the suspension medium and photochemical release of ATP activates only those pump molecules which have the ATPbinding site facing outward.…”

Section: Fluorescence Signals From Reconstituted Vesiclesmentioning

confidence: 99%

Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements

et al. 1991

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Membrane fragments containing a high density of Na,K-ATPase can be noncovalently labeled with amphiphilic styryl dyes (e.g., RH 421). Phosphorylation of the Na,K-ATPase by ATP in the presence of Na+ and in the absence of K+ leads to a large increase of the fluorescence of RH 421 (up to 100%). In this paper evidence is presented that the styryl dye mainly responds to changes of the electric field strength in the membrane, resulting from charge movements during the pumping cycle: (i) The spectral characteristic of the ATP-induced dye response essentially agrees with the predictions for an electrochromic shift of the absorption peak. (ii) Adsorption of lipophilic anions to Na,K-ATPase membranes leads to an increase, adsorption of lipophilic cations to the decrease of dye fluorescence. These ions are known to bind to the hydrophobic interior of the membrane and to change the electric field strength in the boundary layer close to the interface. (iii) The fluorescence change that is normally observed upon phosphorylation by ATP is abolished at high concentrations of lipophilic ions. Lipophilic ions are thought to redistribute between the adsorption sites and water and to neutralize in this way the change of field strength caused by ion translocation in the pump protein. (iv) Changes of the fluorescence of RH 421 correlate with known electrogenic transitions in the pumping cycle, whereas transitions that are known to be electrically silent do not lead to fluorescence changes. The information obtained from experiments with amphiphilic styryl dyes is complementary to the results of electrophysiological investigations in which pump currents are measured as a function of transmembrane voltage. In particular, electrochromic dyes can be used for studying electrogenic processes in microsomal membrane preparations which are not amenable to electrophysiological techniques.

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Section: Fluorescence Signals From Reconstituted Vesiclesmentioning

confidence: 99%

Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements

et al. 1991

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“…Artificial minicells consist of predominantly single‐walled liposomes containing purified Na,K‐ATPase molecules inserted randomly in their membranes (F. 1A); they contain a reservoir of ATP and Na for activating the right‐side‐oriented Na,K‐ATPase upon external Rb or K addition 8 . When ouabain is added to artificial minicells which are in the process of Rb accumulation, the uptake is interrupted 1 and the Rb ions remain entrapped throughout a 15‐minute gel‐filtration step despite an inside‐out Rb gradient, 8 indicating that no Rb ions leak across the E 2 ‐ouabain form.…”

Section: Resultsmentioning

confidence: 99%

“…1A); they contain a reservoir of ATP and Na for activating the right‐side‐oriented Na,K‐ATPase upon external Rb or K addition 8 . When ouabain is added to artificial minicells which are in the process of Rb accumulation, the uptake is interrupted 1 and the Rb ions remain entrapped throughout a 15‐minute gel‐filtration step despite an inside‐out Rb gradient, 8 indicating that no Rb ions leak across the E 2 ‐ouabain form. The gel filtration procedure used for removal of external isotope is a sensitive indicator of pump tightness 9 .…”

Section: Resultsmentioning

confidence: 99%

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Transposing Results from an Artificial Minicell to a Real Cell

Anner

1997

Annals of the New York Academy of Sciences

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The present work sets in parallel data obtained with the Na,K-ATPase in artificial vesicular membranes (artificial minicells) and in peripheral human lymphocytes ex vivo. The Na,K-ATPase was purified and reconstituted into single-walled, tight liposomes filled with a reservoir of ATP and Na in which the Na,K-ATPase functions as in cells, that is, the receptor is accessible on the liposome surface (artificial minicells). In this system, an E2-ouabain state impermeable to Rb ions and an Na,K-ATPase-palytoxin state leaky for Rb ions were characterized. The tight E2-ouabain form preserves the viability of isolated lymphocytes, whereas the leaky Na,K-ATPase-palytoxin induces rapid cell bursting and death by a ouabain-sensitive mechanism. Thus, the effect of Na,K-ATPase inhibitors on lymphocyte survival can be predicted from permeability measurements in artificial minicells as verified also with the leak-inducing metals mercury and silver.

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Reconstitution of ion-motive transport ATPases in artificial lipid membranes

Villalobo¹

1990

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Characterization of (Na+ + K+)-ATPase-liposomes. III. Controlled activation and inhibition of symmetric pumps by timed asymmetric ATP, RbCl, and cardiac glycoside addition

Cited by 10 publications

References 30 publications

Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements

Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements

Transposing Results from an Artificial Minicell to a Real Cell

Reconstitution of ion-motive transport ATPases in artificial lipid membranes

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