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B M Anner

Publications and source records attributed to B M Anner.

53 records · Page 3Linked to original sources
PubMed1984

Characterization of (Na+ + K+)-ATPase liposomes. I. Effect of enzyme concentration and modification on liposome size, intramembrane particle formation and Na+,K+-transport.

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

Animals↗
PubMed1984

Characterization of (Na+ + K+)-ATPase liposomes. II. Effect of alpha-subunit digestion on intramembrane particle formation and Na+,K+-transport.

The effect of the protein structure of (Na+ + K+)-ATPase on its incorporation into liposome membranes was investigated as follows: the catalytic alpha-subunit of (Na+ + K+)-ATPase was split into low-molecular weight fragments by trypsin treatment and the digested enzyme was reconstituted at the same protein concentration as intact control enzyme. The reconstitution process was quantified by the average number of intramembrane particles appearing on concave and convex fracture faces after freeze-fracture of the (Na+ + K+)-ATPase liposomes. The number of intramembrane particles as well as their distribution on concave and convex fracture faces is not modified by the proteolysis. In contrast, the ATPase activity and the transport capacity of the (Na+ + K+)-ATPase decrease progressively with increasing incubation times in the presence of trypsin and are abolished when the original 100 000 molecular weight alpha-subunit is no longer visible by sodium dodecylsulfate gel electrophoresis. Apparently, functional (Na+ + K+)-ATPase with intact protein structure and digested, non functional enzyme consisting of fragments of the alpha-subunit reconstitute in the same manner and to the same extent as judged by freeze-fracture analysis. We conclude that, while trypsin treatment modifies the (Na+ + K+)-ATPase molecule in a functional sense, it appears not to modify its interaction with the bilayer in producing intramembrane particles. On the basis of our results, we propose a lipid-lipid interaction mechanism for reconstitution of (Na+ + K+)-ATPase.

Animals↗
PubMed1983

A bar model for the pump and channel function of the reconstituted Na+,K+-ATPase.

Purified Na+,K+-ATPase is treated with trypsin. The altered enzyme is then reconstituted into liposomes and the change in active and passive Na+,K+-fluxes is recorded. Trypsin treatment transforms the slow passive Na+,K+-fluxes into leaks. The leak formation is correlated with the degree of proteolysis and the associated decrease in Na+,K+-ATPase activity. The active Na+,K+-transport capacity decreases in parallel with the passive transport. It is thus proposed that the Na+,K+-ATPase molecule primarily contains unspecific transmembrane tunnels that are rendered ion-selective by transverse bars of specific length (bar model).

Animals↗
PubMed1982

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 microprocedure for the preparation of Na,K-ATPase-containing liposomes with a minimal starting material (200 microgram) of purified Na,K-ATPase is presented. Phosphatidylcholine is added gradually to cholate-solubilized Na,K-ATPase of various concentrations and the lipid-induced decrease in enzyme activity is monitored. After removal of the detergent by dialysis, the transport parameters of the resulting Na,K-ATPase-liposomes are established by a microassay. By relating the transport properties to the Na,K-ATPase activity preset before dialysis, a procedure is developed which allows to prepare standardized Na,K-ATPase-liposomes with predictable transport properties.

Adenosine Triphosphate↗
PubMed1981

A transport model for the (Na+/K+)ATPase in liposomes including the (Na+/K+)-channel function.

(Na+/K+)ATPase liposomes of various degrees of reconstitution are formed by varying the amount of phosphatidylcholine added to the soluble (Na+/K+)ATPase before vesicles are formed by cholate removal. In the presence of ATP, the reconstituted sodium pump effectuates (Na+/K+) antiport. In the absence of ATP, the reconstituted sodium pump forms a (Na+/K+) channel. The stable plateaus formed by (1) the active Na+ transport, (2) the active K+ transport, (3) the 'passive' Na+ flux, and (4) the 'passive' K+ flux are determined in the optimally and the partially reconstituted liposomes. The activities of all four vectorial functions vary in a tightly correlated fashion, suggesting that they are mediated by the same transport-active configuration of (Na+/K+)ATPase. A transport model which includes the active and the passive (Na+/K+) fluxes mediated by the sodium pump in liposomes is outlined.

Adenosine Triphosphate↗
PubMed1980

Electron microscopy of phospholipid vesicles reconstituted with purified renal Na,K-ATPase.

Purified Na,K-ATPase after reconstitution into phospholipid vesicles catalyzed an active coupled transport with a ratio close to 3Na/2K. A uniform population of closed vesicles with average diameters close to 900 A are observed after freeze-fracture and thin sectioning. After freeze-fracture intramembranous particles with diameters of 80-100 A are observed. The data suggest that these particles correspond to Na,K-ATPase molecules.

Animals↗
PubMed1980

Reconstitution of the Na+, K+-transport system in artificial membranes.

(1) Purified renal Na+, K+-ATPase was incorporated into phosphatidylcholine-liposomes by using the cholate-dialysis procedure. The passive permeability of the liposomes for Na+ and K+ was determined on 22Na and 86Rb equilibration curves. The rate constant for passive Na-flux was 0.014 min-1 and for K(86Rb)-flux 0.013 min-1. Active transport rates of Na+ and K+ were determined after addition of 3 mM ATP to the liposomes. (2) A micromethod for continuously measuring the ADP production associated with active transport is described. Using this technique, initial velocities of ATP hydrolysis were measured in parallel with Na+-transport and K+-transport. When the Na+-concentration in the incubation medium is increased, the initial velocity of all three processes increase concomitantly. At high turnover, the stoichiometry of coupled Na+, K+-transport catalyzed by the purified reconstituted pump is 2.2Na+: 1K+:1ATP. (3) When the pump has exhausted the vesicular K+-pool, coupled NA+, K+-transport ceases. The pump then carries Na+ uphill without K+-antiport. The Na+: ATP ratio of uncoupled Na+-transport is close to 1:1.

Adenosine Triphosphate↗
PubMed1977

A reconstituted Na+ + K+ pump in liposomes containing purified (Na+ + K+)-ATPase from kidney medulla.

Liposomes containing either purified or microsomal (Na+,K+)-ATPase preparations from lamb kidney medulla catalyzed ATP-dependent transport of Na+ and K+ with a ratio of approximately 3Na+ to 2K+, which was inhibited by ouabain. Similar results were obtained with liposomes containing a partially purified (Na+,K+)-ATPase from cardiac muscle. This contrasts with an earlier report by Goldin and Tong (J. Biol. Chem. 249, 5907-5915, 1974), in which liposomes containing purified dog kidney (Na+,K+)-ATPase did not transport K+ but catalyzed ATP-dependent symport of Na+ and Cl-. When purified by our procedure, dog kidney (Na+,K+)-ATPase showed some ability to transport K+ but the ratio of Na+ : K+ was 5 : 1.

Adenosine Triphosphatases↗
PubMed1977

A possible molecular mechanism of the action of digitalis: ouabain action on calcium binding to sites associated with a purified sodium-potassium-activated adenosine triphosphatase from kidney.

Calcium binding at 0 degrees C to a purified sheep kidney Na+,K+-ATPase was described by linear Scatchard plots. Binding at saturating free calcium was 65-80 nmol/mg of protein, or 30-40 mol of calcium/mol of enzyme. Aqueous emulsions of lipids extracted from Na+,K+-ATPase yielded dissociation constants and maximum calcium-binding values that were similar to those for native Na+,K+-ATPase. Phospholipase A treatment markedly reduced calcium binding. Pretreatment of native Na+,K+-ATPase with ouabain increased the dissociation constant for calcium binding from 131 +/- 7 to 192 +/- 7 muM without altering maximum calcium binding. Ouabain pretreatment did not affect calcium binding to extracted phospholipids, ouabain-insensitive ATPases, or heat denatured Na+,K+-ATPase, Na+ and K+ (5-20 mM) increased the dissociation constants for calcium, which suggests competition between the monovalent cations and calcium for the binding sites. At higher concentrations of monovalent cations, ouabain increased the apparent affinity of binding sites for calcium. Extrapolation to physiological cation concentrations revealed that the ouabain-induced increase in apparent affinity for calcium may be as much as 2- to 3-fold. These results suggest: (1) calcium binds to phospholipids associated with Na+,K+-ATPase; (2) ouabain interaction with Na+,K+-ATPase induces a perturbation that is transmitted to adjacent phospholipids, altering their affinity for calcium; and (3) at physiological concentrations of Na+ or K+, or both, ouabain interaction with Na+,K+-ATPase may lead to an increased pool of membrane-bound calcium.

Adenosine Triphosphatases↗
PubMedDate not supplied

Na,K-ATPase characterized in artificial membranes. 1. Predominant conformations and ion-fluxes associated with active and inhibited states.

The Na,K-ATPase (NKA) system is the receptor for the cardioactive steroids of plant or animal origin. It is not yet known whether passive ion fluxes traverse the inactivated receptor and thereby contribute to the hormonal, pharmacological or toxic actions of these compounds. To look for putative passive ion-fluxes across the ouabain-NKA complex, we incorporated it into the artificial membrane of liposomes. Since this synthetic membrane is virtually impermeable to Na and K ions, the hypothetical ion-fluxes mediated by the NKA can be determined. E2-forms and E2-ouabain-forms of purified NKA were incorporated, in parallel, into separate liposome preparations and the permeability of the resulting E2-liposomes and E2-ouabain-liposomes to K, Na and Ca ions was compared. The E2-liposomes expressed a typical K-permeability which was not observed in the E2-ouabain-liposomes; the latter showed a slightly higher Na-permeability and a similar Ca-permeability as compared to the former. Thus, ouabain does not induce leaks for K or Ca ions in the NKA molecule.

Adenosine Triphosphate↗
PubMedDate not supplied

Na,K-ATPase characterized in artificial membranes. 2. Successive measurement of ATP-driven Rb-accumulation, ouabain-blocked Rb-flux and palytoxin-induced Rb-efflux.

The Na,K-ATPase is a multifunctional system anchored in the membrane of eukaryotic cells; it is responsible for the establishment and regulation of the Na/K balance of cell and organism by a stoichiometric mechanism linking Na extrusion to K uptake and ATP hydrolysis. The receptor for cardioactive steroids such as digoxin and ouabain is located at the extracellular surface of the system. Conversely, palytoxin, the most potent animal toxin, exerts its toxic effect by creating nonspecific leaks in the cell membrane leading to K-efflux and influx of Na and Ca ions. Ouabain prevents the pore-forming action of palytoxin in cells and therefore Na,K-ATPase is suspected to be the common receptor of ouabain and palytoxin. We have developed an artificial membrane system to determine structure-function relationships and ligand interactions of purified Na,K-ATPase: two-sided, bi-directional ATP-filled liposomes. In this system, ATP-driven 86Rb accumulation, arrest of 86Rb-uptake by ouabain, and palytoxin-induced 86Rb-leak were measured successively in the same preparation. Ouabain prevented the leak when the enzyme was ouabain-sensitive (rabbit kidney) but not when it was ouabain-resistant (rat kidney). On the basis of these data in conjunction with conformational analyses, allosteric conformational competition for the ouabain-palytoxin antagonism is proposed.

Acrylamides↗
PubMedDate not supplied

Two brain inhibitors inhibit renal Na,K-ATPases without recognizing the species-dependent variation of their ouabain-sensitivity.

The membrane Na,K-ATPase is the driving force for sodium reabsorption in the kidney. Accordingly, Na,K-ATPase has been proposed to be a likely target for the action of a putative natriuretic hormone which would modulate sodium excretion by partial inhibition of renal Na,K-ATPase activity. To examine this hypothesis, it is necessary to isolate inhibitors from body fluids and tissues and to characterize their interaction with Na,K-ATPase in comparison to the plant inhibitors ouabain. Two inhibitors extracted from hypothalamus or hypothalamus-hypophysis have been compared to ouabain with regard to the shape of the dose-response curves and species-dependence. Ouabain inhibited renal Na,K-ATPase with dose-response curves spanning 3 to 5 orders of magnitude and marked species-dependence. By contrast, the brain inhibitors blocked the ATPase activity of isolated renal Na,K-ATPase with steep dose-response curves without species-dependence. Thus, the brain inhibitors are clearly distinct from plant ouabain; their chemical structures remain to be established.

Animals↗