Use of two-sided bifunctional liposomes in the study of a hypothalamic Na,K-ATPase inhibitor (original) (raw)

Right-side-out pumping Na,K-ATPase-liposomes: a new tool to study the enzyme's receptor function

The technology to prépare right-side-out pumping Na,K-ATPaseliposomes is described. The 50% right-side-out oriented pumps of ATP-containing liposomes are then activated by thé addition of external Rb ions, leading to a ouabain-sensitive Rb-influx which is thé mirror-image of thé inside-out transport. The resulting internai Rb concentration is 4 to 10 fold larger than thé external concentration. Finally, thé accumulated Rb ions can be extruded by driving thé 50% inside-out oriented pumps by external ATP.

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

Biochimica Et Biophysica Acta-biomembranes, 1987

Inside-out as welI as right-side-out oriented (Na"^ + K"^)-ATPase molécules reconstituted in liposomes are activated successively by timed asymmetric addition of ATP to the internai and extemal liposome compartment; this présents the first functional confirmation of the symmetric pump-orientation in cholatedialysed préparations revealed previousiy by the equal distribution of intramembrane particles on the concave and convex surface of freeze-fractured (Na"^ + K )-ATPase-liposomes. The initial transport rates of the symmetrically oriented pump populations are regulated by varying the bilatéral K or Rb ion concentrations; ATP, ouabain, digoxin or vanadate are used to activate or block selectively the right-side-out, inside-out or both (Na^-I-K•^)-ATPase populations. Finally, thèse liposomes of the second génération présent a new tool to evaluate the membrane-permeability as well as the effects of receptor-ligands or other probes in a single préparation.

A reconstituted Na,K-pump in liposomes containing purified Na,K-ATPase from kidney medulla

The membrane transport system for Na and K ions was isolated and inserted in a functional state into artifical nanovesicles, which form spontaneously by fusion of microdiscs containing the Na,K-ATPase molecules - upon detergent removal. Na and K are exchanged without back-leaks despite a steep uphill gradient. The uphill ion fluxes are blocked by occupation of the receptor site of the system by ouabain - without leak formation, just by arrest of ion-pump turnover.

CHARACTERIZATION OF (Na+ + K + )-ATPase LIPOSOMES I. EFFECT OF ENZYME CONCENTRATION AND MODIFICATION ON LIPOSOME SIZE, INTRAMEMBRANE PARTICLE FORMATION AND Na+,K+-TRANSPORT

Rabbit rénal (Na++ K+)-ATPase (EC 3.6.1.3) was purified and incorporated into phosphatidylcholine liposomes. Freeze-fracture analysis of thé reconstituted System reveals intramembrane particles formed by (Na+ + K + )-ATPase molécules which are randomly distributed on concave and convex fracture faces. The reconstituted (Na+ + K+)-ATPase performs active Na+,K +-transport. The distribution of particles as well as thé rate of active transport are directly proportional to thé (Na+ + K + )-ATPase protein concentration used for reconstitution, while thé total amount of sodium and potassium ions exchanged by ATP per volume vesicle suspension reaches maximum when each vesicle contains on thé average more than two particles. (Na+ + K + )-ATPase pretreated with ouabain or vanadate yields thé same particle density and vesicle size as control enzyme. However, detergent-denatured enzyme loses its ability to form intramembrane particles or to increase thé vesicle size indicating that thé lipids surrounding thé protein part of thé molécule are essential for thé reconstitution process. The vesicle diameter increases as a function of thé number of particles per vesicle. Histograms of thé size distribution become wider with increasing intramembrane particle density and tend to show more than one maximum.

Preparation of Na,K-ATPase-containing liposomes with predictable transport properties by a procedure relating the Na,K-transport capacity to the ATPase activity

Journal of Biochemical and Biophysical Methods, 1982

A mieroprocedure for thé préparation of Na.K-ATPase-containing, liposomes with a minimal starting material (200 /ig) of purificd Na,K-ATPa,se is présentée!. Phosphatidylcholine is addcd gradually to cholate-solubilizcd Na.K-ATPase of various concentrations and thé lipid-induced decrcase in enzyme activity is monitorcd. After removal of thé détergent hy dialysis, thc Iransport paramciers of thé resulting Na.K-ATPasc-liposomes are established by a microassay. By relating thé transport properties to thé Na,K-ATPase activity présent beforc dialysis, a procédure is devclopcd which allows to prépare standardizcd Na.K-ATPase-liposomcs with predictable transport properties.