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B M Anner

Publications and source records attributed to B M Anner.

At least 37 records · Page 2Linked to original sources
PubMed1992

Mercury blocks Na-K-ATPase by a ligand-dependent and reversible mechanism.

An inhibitory receptor for cardioactive steroids such as digoxin and ouabain is located at the extracellular surface of the Na-K-adenosinetriphosphatase (ATPase) molecule. Besides cardioactive steroids, mercury is a potent inhibitor of the Na-K-ATPase activity. The half-maximal inhibitory concentration (IC50), determined within 30 min at 37 degrees C at 1 microgram protein/ml, was 200 nM, despite the presence of 1 mM EDTA; the IC50 decreased with increasing protein/inhibitor ratio, and it reached 2.7 microM at 0.1 mg protein/ml and 20 microM at 1 mg protein/ml. The IC50 for Na-K-ATPase inhibition by the diuretic compound mersalyl was 4 and 5 microM for the nondiuretic p-chloromercuribenzenesulfonic acid at 0.1 mg protein/ml. The IC50 for HgCl2 inhibition was modulated by the presence of EDTA as well as by the pump ligands Mg, Na, K, and ATP. The E2 conformation of the Na-K-ATPase molecule was more sensitive to HgCl2 than the E1 conformation. The mercury antidote 2,3-dimercapto-1-propanesulfonic acid was able to reactivate approximately 70% of the blocked enzyme. In conclusion, a metal-binding domain of the Na-K-ATPase molecule with particular high affinity for Hg(II) was described functionally in the present work. Therefore Na-K-ATPase belongs to the metal-binding proteins. Metals may modulate the cellular expression and activity of the system by interacting with its metal-binding interface.

Adenosine Triphosphate↗
PubMed1992

Mercury weakens membrane anchoring of Na-K-ATPase.

The presence of circulating inhibitors able to decrease the renal Na-K-adenosinetriphosphatase (ATPase) activity (natriuretic hormones) was postulated some 30 years ago. In the present work, the natriuretic inhibitor HgCl2 was selected as a model compound for the structural characterization of a possible natriuretic pathway for Na-K-ATPase modification. The structural effects of Na-K-ATPase inhibition by HgCl2 were assessed by trypsinolysis of the blocked enzyme in comparison with untreated preparations. The results show that inactivation of Na-K-ATPase by HgCl2 leads to the release of the alpha-subunit from the membrane preferentially in the E2 conformation but also in the E1 conformation. Apparently, HgCl2 weakens the membrane anchoring of the alpha-subunit, presumably by loosening the alpha-beta-subunit interaction. By this mechanism, the sensitivity of the Na-K-ATPase to extracellular drugs, hormones, and antibodies, as well as to intracellular proteases and other regulatory factors, could be altered.

Animals↗
PubMed1992

Mercury inhibits Na-K-ATPase primarily at the cytoplasmic side.

The investigation of active Na-K transport inhibition by mercury is difficult to perform in a cell because of the presence of numerous other membrane and intracellular proteins modifiable by mercury. Thus purified Na-K-adenosinetriphosphatase (ATPase) molecules performing active transport in an artificial membrane are required to demonstrate unequivocally the inhibition of active transport by mercury. We made use of a single population of Na-K-ATPase liposomes filled with ATP and Na to show mercury inhibition of active 86Rb transport mediated by both the inside-out and right-side-out pumps in the same liposome. The effect of HgCl2 on the Na-K-ATPase in cell-like and reversed orientation was measured in comparison with convallatoxin. A dilution series showed that 10 microM externally added HgCl2 inhibited the active 86Rb transport at the cytoplasmic side first; at 50 microM both pump populations were blocked, indicating either membrane permeation by HgCl2 and inhibition at the internal intracellular domains or onset of extracellular action at higher HgCl2 concentration. The results show that the metal-binding interface of Na-K-ATPase molecule is profoundly implicated in active ion transport and that the intracellular part of the Na-K-ATPase molecule presents the primary target for mercury action.

Biological Transport, Active↗
PubMed1990

Chelation of mercury by ouabain-sensitive and ouabain-resistant renal Na,K-ATPase.

The SH-reactive HgCl2 inhibits the Na,K-ATPase activity potently in a manner antagonized only partially by EDTA or cysteine; solely dimercaprol, a dithiol antidote for mercury, blocks the HgCl2 effects entirely as confirmed also by 203Hg-binding experiments. The results reveal the presence of a chelating component in pure Na,K-ATPase with an affinity for mercury superior to EDTA. The mercury-sensitivity of the Na,K-ATPase is not related to the ouabain-sensitivity. This criterion will be useful for the distinction between ouabain-like and mercury-like inhibitors from body fluids and tissues.

Animals↗
PubMed1990

Hypothalamic Na(+)-K(+)-ATPase inhibitor characterized in two-sided liposomes containing pure renal Na(+)-K(+)-ATPase.

The functional characterization of putative endogenous inhibitors of the Na(+)-K(+)-ATPase has been greatly hindered by spare amounts extractable from biological sources. We therefore used a miniaturized, two-sided test system consisting of ATP-filled liposomes containing dispersed, randomly oriented renal Na(+)-K(+)-ATPase molecules to study effects of a low-molecular-weight, nonpeptidic Na(+)-K(+)-ATPase inhibitor extracted from bovine hypothalamus. With this test system, Na(+)-K(+)-ATPase inhibition produced by a single dose of 0.1 U (congruent to 75 fmol) of the hypothalamic inhibitory factor (HIF) as well as the membrane permeation of a single unit (approximately equal to 750 fmol) became measurable, and an estimation of the minimal number of HIF molecules per unit could be made. By a molecular mechanism involving positive cooperativity, HIF potently and completely blocked active 86Rb+ transport catalyzed by the right-side-out-oriented pump population, with an average 50% inhibitory concentration of 3.5 x 10(-8) M, indicating a roughly 30-fold higher apparent affinity than ouabain. By studying inhibition of the inside-out-oriented pump population, comparison of the membrane permeability of HIF to that of various cardiac glycosides of known hydrophobicity further indicated that HIF is not entirely ouabain-like as HIF penetrates the liposomal membrane, whereas ouabain does not. Besides the cardiac glycosides, HIF is the only compound tested thus far in the purified system that displays such striking transport inhibition. Other known or proposed endogenous Na(+)-K(+)-ATPase inhibitors, including unsaturated fatty acids, palytoxin, dehydroepiandrosterone, and vanadate, produce only partial transport inhibition even at high concentration.

Animals↗
PubMed1989

Sodium transport defect of ouabain-resistant renal Na,K-ATPase.

The murine renal Na,K-ATPase is resistant to cardiac glycosides. It is not yet known however whether altered active transport is associated with the drug-resistance. To investigate this problem Na,K-ATPases were purified from the outer medulla of both rat and rabbit kidneys and reconstituted identically into liposomes. The Na-stimulation of the Na,K-ATPase activity before reconstitution and of the Na-transport after reconstitution was measured. A Na-defect inherent in the ouabain-resistant rat Na,K-ATPase was discovered indicating a link between the cardiac glycoside sensitivity and the Na-transport.

Animals↗
PubMed1987

[Digitalis receptor].

It has been well established that the Na+,K+-ATPase is the pharmacological receptor of cardiac glycosides. One or several endogenous analogues of these digitalis compounds have been shown to interfere with radioimmunoassays (RIA). Given the low therapeutic range of the steroid cardiotonics, such interference must be taken into consideration. The present review briefly describes the digitalis receptor and discusses monitoring by RIA of patients treated with cardiac glycosides. Finally, the putative origin and chemical nature of this (these) endogenous digoxin-like factor(s) are presented in detail.

Adult↗
PubMed1987

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

Inside-out as well as right-side-out oriented (Na+ + K+)-ATPase molecules reconstituted in liposomes are activated successively by timed asymmetric addition of ATP to the internal and external liposome compartment; this presents the first functional confirmation of the symmetric pump-orientation in cholate-dialysed preparations revealed previously 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 bilateral 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+ + K+)-ATPase populations. Finally, these liposomes of the second generation present a new tool to evaluate the membrane-permeability as well as the effects of receptor-ligands or other probes in a single preparation.

Adenosine Triphosphate↗
PubMed1985

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

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

Adenosine Triphosphate↗
PubMed1985

Optical study of active ion transport in lipid vesicles containing reconstituted Na,K-ATPase.

A fluorescence method is described for the measurement of ATP-driven ion fluxes in lipid vesicles containing purified Na,K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made selectively permeable to K+ so that the membrane voltage approaches the Nernst potential for K+. With constant external K+ concentration, the time course of internal K+ concentration can be continuously measured as change of the fluorescence signal after activation of the pump. The optical method has a higher time resolution than tracer-flux experiments and allows an accurate determination of initial flux rates. From the temperature dependence of active K+ transport its activation energy was determined to be 115 kJ/mol. ATP-stimulated electrogenic pumping can be measured as fast fluorescence change when the membrane conductance is low (i.e., at low or zero valinomycin concentration). In accordance with expectation, the amplitude of the fast signal change increases with decreasing passive ion permeability of the vesicle membrane. The resolution of the charge movement is so high that a few pump turnovers can be easily detected.

Animals↗
PubMed1984

Leakage-channel conductance of single (Na+ + K+)-ATPase molecules incorporated into planar bilayers by fusion of liposomes.

By fusing liposomes which contain in the mean only one pump unit (one intramembranous particle) to planar bilayers, and provoking the ouabain-blockable leakage conductance by the presence of n-decane, the predominant unit leakage conductance associated with one pump unit was estimated to be 40-50 pS, indicating the channel nature of the leakage pathway.

Electric Conductivity↗