Affinity For Ouabain Research Articles

Sodium load and high affinity ouabain binding in rat and guinea-pig cardiac tissue.

An estimation of the actual Na/K-ATPase transport activity in intact cardiac cells was made by measuring the binding of [3H]-ouabain to rat and guinea-pig ventricular strips. At the low [3H]-ouabain concentration of 1 nM equilibrium binding was hardly obtained after an incubation time of five hours. Different procedures known to alter the sodium load of the cardiac preparations influenced [3H]-ouabain binding: the sodium ionophore monensin enhanced [3H]-ouabain binding, the local anaesthetic dibucaine and a reduction of external sodium ion concentration diminished [3H]-ouabain binding; [3H]-ouabain binding was similarly affected by these procedures in the rat and guinea-pig. Since [3H]-ouabain binding occurred predominantly at the high-affinity binding sites of rat myocardium under the applied experimental conditions, it was concluded that these binding sites represent Na/K-ATPase molecules involved in sodium ion transport.

Characterizing osmotically-induced Na,K-ATPase from Callinectes sapidus

Blue crabs, Callinectes sapidus, when Actapted to low salinity show an increase in gill Na,K-ATPase that: 1. 1. is maximal on day 7 and is confined to gills 6 and 7; 2. 2. is sedimented at 9500 g but is not accompanied by a corresponding increase in Mg-ATPase; 3. 3. has an altered affinity for ouabain.

Digitalis receptors in cardiac cells and their relation with positive inotropic and cardiotoxic effects.

Biochemical and pharmacological properties of (Na+,K+)ATPase have been studied on intact chick embryonic hearts and under heart cell culture conditions. The results show the existence of two families of ouabain binding sites: a low affinity binding site with a dissociation constant (Kd) of 2-6 microM for the ouabain-receptor complex and a high affinity binding site with a Kd of 26-48 nM. High and low affinity sites are also present at all embryonic stages studied. Inhibition of 86Rb+ uptake in cultured cardiac cells and increase in intracellular Na+ concentration, due to (Na+,K+)ATPase blockade, occur in a ouabain concentration range corresponding to the saturation of the low affinity ouabain site. Ouabain stimulated 45Ca2+ uptake increases in parallel with the increase in the intracellular Na+ concentration. It is suppressed in Na+ free medium or when Na+ is replaced by Li+ suggesting that the increase is due to the indirect activation of the Na+/Ca2+ exchange system in the plasma membrane. Dose-response curves for the inotropic effects of ouabain on papillary muscle and on ventricular cells in culture indicate that the development of the cardiotonic properties is parallel to the saturation of the low affinity binding site for ouabain. Therefore, inhibition of the cardiac (Na+,K+)ATPase corresponding to low affinity ouabain binding sites seems to be responsible for both the cardiotonic and cardiotoxic effects of the drug.

Effects of myocardial hypoxia on digitalis-induced toxicity in the isolated heart of guinea pigs and cats

The effects of hypoxia on the tolerance of myocardium to the toxic actions of digitalis were studied in isolated heart muscle preparations. Perfusion of Langendorff preparations of guinea-pig heart with a hypoxic solution failed to alter Na, K-ATPase with respect to its activity, its sensitivity to inhibition by dihydrodigoxin, or the number of [ 3H]ouabain binding sites and their affinity for ouabain, when these activities were estimated in ventricular muscle homogenates obtained after the hypoxic perfusion. Hypoxia potentiated the development of digoxin-induced contracture, as well as digoxin-induced changes in maximal upstroke velocity and the amplitude of action potentials, and the resting transmembrane potential in atrial and ventricular muscle preparations of guinea-pig heart. Under hypoxic conditions, however, digoxin failed to induce arrhythmias. In Purkinje fibers of cat heart, the time to onset of digoxin-induced arrhythmias, or the development of afterdepolarizations, was not affected by hypoxia. These results indicate that hypoxia alters the nature of toxic effects of digitalis in isolated heart muscle preparations, the development of contracture instead of arrhythmias being the primary toxicity in hypoxic muscle.

Characterization of Na+, K+‐ATPase in Cultured and Separated Neuronal and Glial Cells from Rat Cerebellum

The activities of certain properties of sodium, potassium-activated adenosine triphosphatase (Na+,K+-ATPase; EC 3.6.1.3) were examined in cultures and perikarya fractions enriched in rat cerebellar nerve cells or astrocytes, in comparison with preparations from whole immature and adult rat cerebellum and derived synaptosomal fractions, as well as nonneural tissue such as the kidney. The specific activity of Na+,K+-ATPase was markedly higher in the freshly isolated astrocytes than in the nerve cells (3-15-fold greater depending on neuronal cell type). In contrast, the specific activity of the enzyme was about twice as high in the primary neuronal as in the astrocytic cultures after 14 days in vitro. In membrane preparations from the whole cerebellum, synaptosomal fractions, and total perikarya suspensions the inhibition of enzyme activity by ouabain indicated complex kinetics, which were consistent with the presence of two forms of the Na+,K+-ATPase (apparent Ki values of about 10(-7) M and 10(-4)-10(-5) M, respectively), the high-affinity form accounting for 60-75% of the total activity. The interaction of the enzyme with ouabain was apparently similar in perikarya preparations of granule neurones, Purkinje cells, and astrocytes. Differences were, however, observed in the properties of the Na+,K+-ATPase of cultured neurones and astrocytes. The latter contained predominantly, but not exclusively, an Na+,K+-ATPase with low affinity for ouabain (73% of the total) that is similar to the single enzyme form in the kidney. This form constituted a significantly smaller proportion of the Na+,K+-ATPase in the cultured neuronal preparations (55%). It would appear, therefore, that in membrane fractions from preparations enriched in different separated and cultured neural cell types both the high- and the low-affinity forms of the enzyme, in terms of interaction with ouabain, are expressed. Depending on the class of cells these enzyme forms constituted a different proportion of the total activity, but both forms seemed to be present in every type of cell examined, even after taking into account the contribution in the enriched preparations of the contaminating cell types. In contrast with the results on the Na+,K+-ATPase activity determined under optimal conditions in preparations derived from disrupted cells, differences could not be detected between the cultured cell types when the effect of ouabain on the uptake of 86Rb into "live cells" was estimated as a measure of in situ ion pump activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Digitalis receptors affinity labelling and relation with positive inotropic and cardiotoxic effects.

Affinity labelling of the digitalis receptor has indicated that it is situated on the N-terminal part of the alpha-subunit of the (Na+,K+)ATPase. Biochemical and pharmacological properties of the (Na+,K+)ATPase studied on intact chick embryonic hearts and under heart cell culture conditions have indicated the existence of two families of ouabain binding sites i.e.: a low affinity binding sites with a dissociation constant (Kd) of 2-6 microM for the ouabain-receptor complex and a high affinity binding site with a Kd of 26-48 nM. High and low affinity sites also are present at all embryonic stages studied. Inhibition of 86Rb+ uptake in cultured cardiac cells and increase in intracellular Na+ concentration, due to (Na+,K+)ATPase blockade, occur in an ouabain concentration range corresponding to the saturation of the low affinity ouabain site. Ouabain stimulated 45Ca2+ uptake increases in parallel with the increase in the intracellular Na+ concentration. It is suppressed in Na+ free medium or when Na+ is replaced by Li+ suggesting that the increase is due to the indirect activation of the Na+/Ca2+ exchange system in the plasma membrane. Dose-response curves for the inotropic effects of ouabain on papillary muscle and on ventricular cells in culture indicate the development of the cardiotonic properties is parallel to the saturation of the low affinity binding site for ouabain. Therefore, inhibition of the cardiac (Na+,K+)ATPase corresponding to low affinity ouabain binding sites seems to be responsible for both the cardiotonic and cardiotoxic effects of the drug.

Two ouabain binding sites in guinea pig heart Na+-K+-ATPase. Differentiation by sodium and erythrosin B.

3H-ouabain binding to guinea pig heart NaK-ATPase resulted in two different cardiac glycoside binding sites: A high affinity, low capacity binding was obtained at a KD = 2.6 X 10(-7) mol/l (about 40% of the binding sites) whereas a low affinity, high capacity binding was established at a KD = 1.3 X 10(-6) mol/l (about 60% of the binding sites). Similar results were obtained when studying the effect of increasing Na+-concentrations on ouabain binding: again two distinct processes involved in the Na+/ouabain interaction could be demonstrated. Whereas one ouabain binding process was activated at rather low Na+-concentrations (K0.5 = 4.5 mmol/l), the other ouabain binding process was predominant at high Na+-concentrations only (K0.5 = 69 mmol/l). Comparing these data with the ouabain action on contractile force and with NaK-ATPase activity in guinea pig heart, the high affinity binding of ouabain seems to correlate with the inotropic action, whereas the low affinity ouabain binding is more related to NaK-ATPase inhibition. To further discriminate the two cardiac glycoside binding sites operative in guinea pig heart muscle, erythrosin B, shown to be an inhibitor of the high affinity ouabain binding in this preparation, was applied to isolated guinea pig left atria in the presence of increasing ouabain concentrations. There was no change in the ouabain induced inotropy or toxicity by erythrosin B. These results could indicate that the low affinity ouabain binding is responsible for both inotropic and toxic actions in guinea pig heart. However, the functional significance of the high affinity ouabain binding would then remain obscure.(ABSTRACT TRUNCATED AT 250 WORDS)

The inotropic effect of ouabain and its antagonism by dihydroouabain in rat isolated atria and ventricles in relation to specific binding sites.

The inotropic effect of ouabain has been studied in rat ventricles and atria. The concentration-effect curve of ouabain may be fitted by a model assuming the existence of two saturable components. The component with the higher sensitivity to ouabain accounted for 30% of the maximal increase in systolic tension in ventricles and for only 5% in atria. Increase in diastolic tension was only apparent at ouabain concentrations required to observe the low sensitivity component. [3H]-ouabain binding has been examined in microsomes prepared from atria and ventricles. High and low affinity binding sites have been observed. The ratio of high and low affinity ouabain binding sites was 4 fold lower in microsomes from rat atria than from rat ventricles. This could account for the difference in the response of these two tissues to the inotropic action of ouabain. In ventricular strips the high sensitivity component was much less apparent in the presence of dihydroouabain than with ouabain. When ventricular strips were preincubated in the presence of dihydroouabain 3 microM, the increase in systolic tension evoked by ouabain 1 microM was significantly reduced. Cumulative concentration-effect curve studies showed dihydroouabain antagonism to the high sensitivity component.

Two classes of ouabain receptors in chick ventricular cardiac cells and their relation to (Na+,K+)-ATPase inhibition, intracellular Na+ accumulation, Ca2+ influx, and cardiotonic effect.

The biochemical and pharmacological properties of the (Na+,K+)-ATPase have been studied at different stages of chick embryonic heart development in ovo and under cell culture conditions. The results show the existence of two families of ouabain binding sites: a low affinity binding site with a dissociation constant (Kd) of 2-6 microM for the ouabain-receptor complex and a high affinity binding site with a Kd of 26-48 nM. Levels of high affinity sites gradually decrease during cardiac ontogenesis to reach a plateau near 14 days of development. Conversely the number of low affinity binding sites is essentially invariant between 5 days and hatching. Cultured cardiac cells display the same binding characteristics as those found in intact ventricles. Inhibition of 86Rb+ uptake in cultured cardiac cells and an increase in intracellular Na+ concentration, due to (Na+,K+)-ATPase blockade, occur in a ouabain concentration range corresponding to the saturation of the low affinity ouabain site. Ouabain-stimulated 45Ca2+ uptake increases in parallel with the increase in the intracellular Na+ concentration. It is suppressed in Na+-free medium or when Na+ is replaced by Li+ suggesting that the increase is due to the indirect activation of the Na+/Ca2+ exchange system in the plasma membrane. Dose-response curves for the inotropic effects of ouabain on papillary muscle and on ventricular cells in culture indicate that the development of the cardiotonic properties is parallel to the saturation of the low affinity binding site for ouabain. Therefore, inhibition of the cardiac (Na+,K+)-ATPase corresponding to low affinity ouabain binding sites seems to be responsible for both the cardiotonic and cardiotoxic effects of the drug.

Influence of cationic amphiphilic drugs on the characteristics of ouabain-binding to cardiac Na +/K +-ATPase

The influence of 12 cationic amphiphilic compounds on the equilibrium and kinetic characteristics of the binding of tritium-labelled ouabain to the lipoprotein Na +/K +-ATPase present in a crude membrane suspension of guinea pig myocardium was investigated. The drugs, e.g. local anaesthetic, antiarrhythmic and psychotropic agents, inhibited specific binding of ouabain in a concentration-dependent manner by reducing its affinity without affecting the number of binding sites. In the presence of chlorpromazine, propranolol and dibucaine, the decreased affinity of ouabain was due to both a diminished association rate and an increased dissociation rate, while in the presence of the weakly potent procaine only the association rate of ouabain was found to be reduced. The different potency of the catamphiphilic drugs was well correlated to the degree of their hydrophobicity. Evidence is presented that the protonized form of the drugs is the effective one. Concerning the mode of action, the catamphiphilic drugs are proposed to interact with the phospholipid part of the lipoprotein Na +/K +-ATPase, thereby indirectly altering the conformation of the embedded protein moiety and thus reducing the proper fit between ouabain and its receptor.

Phosphorylation of the (Na,K)-ATPase by a plasma membrane-bound protein kinase in friend erythroleukemia cells.

A decrease in the activity of the (Na,K)-ATPase is an early and essential step in commitment of Friend virus-infected murine erythroleukemia cells to terminal erythroid differentiation. Plasma membranes from these cells were purified and shown to contain ouabain-inhibitable (Na,K)-ATPase present as approximately 0.4% of the total membrane protein. Protein kinase activity also co-purified with the plasma membrane and preferentially phosphorylated a Nonidet P-40 detergent-extractable 100,000-Da peptide. The 100,000-Da phosphopeptide migrated with the alpha subunit of dog kidney (Na,K)-ATPase when electrophoresis was carried out in the presence of sodium dodecyl sulfate in either 5 or 10% polyacrylamide gels. In two-dimensional gel electrophoresis, it separated into a series of spots between pH 5.1 and 5.4, while dog kidney alpha subunit appeared as a doublet at pH 5.3-5.4. When Nonidet P-40-solubilized plasma membranes were passed through a ouabain affinity column in the presence of Mg2+, Na+, and ATP, the 100,000-Da phosphopeptide was retained and could be eluted by ouabain. This peptide was also phosphorylated in living murine erythroleukemia cells, and proteolysis patterns of the peptide labeled in vitro, the peptide labeled in vivo, and the purified dog kidney alpha subunit using V8 protease were nearly identical. Phosphothreonine was detected in both the peptides labeled in vivo and in vitro.

Changes in the number and activity of sodium pumps in erythrocytes from patients with hyperthyroidism.

1. The sodium content, the rate and rate constant of ouabain-sensitive sodium efflux and the number of sodium pumps (indicated by the ouabain-binding capacity) were measured in erythrocytes from patients with hyperthyroidism and compared with values obtained in euthyroid patients and healthy control subjects. Erythrocyte zinc content was measured as a simple estimate of the content of carbonic anhydrase. 2. In the hyperthyroid patients, erythrocyte sodium content was increased, whereas the rate and rate constant of ouabain-sensitive sodium efflux, the ouabain-binding capacity and erythrocyte zinc content were all decreased. 3. The sodium pumps in hyperthyroidism had the same affinity for ouabain and the same rate constant per pump as those in healthy controls. 4. The decrease in the efflux rate constant could be entirely accounted for by the decrease in the number of sodium pumps. 5. Although the sodium efflux was decreased in the hyperthyroid patients, the change was less than expected for the decrease in the efflux rate constant. This suggests that there is an increase in the ground permeability of the erythrocyte membrane in hyperthyroidism. 6. In the hyperthyroid patients the number of sodium pumps and erythrocyte zinc content were inversely related to the plasma levels of thyroxine and tri-iodothyronine, but more closely to the latter. 7. These results suggest that the thyroid hormones may influence the erythrocyte's content of a range of proteins which happens to include the sodium pump.

(Na+ + K+)-dependent adenosine triphosphatase. Regulation of inorganic phosphate, magnesium ion, and calcium ion interactions with the enzyme by ouabain.

1. (Na+ + K+)-dependent adenosine triphosphatase was phosphorylated on the alpha-subunit by Pi in the presence of Mg2+. Phosphorylation was stimulated by ouabain. The interactions of Pi, Mg2+, and ouabain with the enzyme could be explained by a random terreactant scheme in which the binding of each ligand to the enzyme increased the affinities for the other two. Dissociation constants of all steps of this scheme were estimated. 2. In the presence of Pi and ouabain and without added Mg2+, the phosphoenzyme was formed. Because this could be prevented by ethylenediaminetetraacetic acid, but not ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, phosphoenzyme formation under these conditions was probably dependent on traces of endogenous Mg2+. The ability of this Mg2+ to support phosphorylation could be explained by the large increase in the enzyme's affinity for Mg2+ by ouabain. 3. In the absence of ouabain, Ca2+ did not support phosphorylation and inhibited Mg2+-dependent phosphorylation. At lower concentrations, Ca2+ was competitive with Mg2+. With increasing Ca2+ concentration, negative cooperativity was observed, suggesting the existence of multiple divalent cation sites with equivalent affinities for Mg2+, but varying affinities for Ca2+. 4. In the presence of ouabain, the maximum inhibition of Mg2+-dependent phosphorylation by Ca2+ was 50%. With saturating Pi, Mg2+, and ouabain, the number of sites binding ouabain was equal to the number of sites phosphorylated. Although Ca2+ halved phosphorylation and reduced the affinity for ouabain about 100-fold, it did not affect the number of ouabain sites. 5. We suggest that the enzyme is an alpha-oligomer and that the half-of-the-sites reactivity for phosphorylation in the presence of Pi, Mg2+, ouabain, and optimal Ca2+ is caused by (a) ouabain-induced increase in the affinities of both protomers for Mg2+ and (b) the inability of Ca2+ to replace Mg2+ on one of the protomers.

Origins of the different sensitivities of [formula omitted] adenosinetriphosphatase preparations to ouabain

1. 1. Properties of the Na +,K +-ATPase preparations from rat and dog kidney medullae were compared. 2. 2. The two enzymes, with a 1000-fold difference in ouabain sensitivities, had similar subunit compositions and similar K 0.5 values and Hill coefficients for substrates and activators of several catalytic activities; suggesting that the structural differences of the two are limited to the ouabain binding domains. 3. 3. Experiments on the interactions of ouabain, Pi, and Mg 2+ with the enzymes showed that the two enzymes differed (a) in their inherent affinities for ouabain; and (b) in that Mg 2+ binding increased affinity for ouabain to a greater extent in the dog enzyme than in the rat enzyme.

Brain (Na +,K +)-ATPase: Biphasic interaction with erythrosin B

Brain (Na +,K +)-adenosine triphosphatase (EC 3.6.1.3) has both high and low affinity ouabain binding sites. It has been proposed that the high affinity ouabain binding sites characterize a nerve-specific form of the enzyme. Erythrosin B has been reported to inhibit high affinity ouabain binding selectively. The experiments in this paper were carried out in order to characterize the interactions of erythrosin B with (Na +,K +)-ATPase and to examine the specificity of erythrosin B for enzyme with high affinity for ouabain. Inhibition by erythrosin B was biphasic, with a rapid and a slow phase. The rapid phase appeared to be relatively specific for enzyme with high affinity for ouabain, while the slow phase was not. Inhibition by erythrosin B was accelerated by Mg 2+ and was retarded by ATP, K +, or Na + and ATP. Erythrosin B increased apparent affinity of the enzyme for K + and decreased apparent affinity for Na + and for ATP. These results indicate that erythrosin B interacts with an ATP site and has effects on cation affinities opposite to those of ATP. Erythrosin B inhibition is proportional to high affinity ouabain binding if brief incubation times and moderate concentrations are used.

The insect brain ( [formula omitted] Binding of ouabain in the hawk moth, Manduca sexta

(1) A quantitative study has been made of the binding of ouabain to the ( Na + + K +)- ATPase in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ( Na + + K +)- ATPase will bind ouabain either in the presence of Mg 2+ and P i, (‘Mg 2+, P i’ conditions) or in the presence of Na +, Mg 2+, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain ( Na + + K +)- ATPase . The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the K D ) of ouabain to the M. sexta brain ( Na + + K +)- ATPase under both binding conditions. However, ouabain binding is more sensitive to K + inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K + inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ( Na + + K +)- ATPase has a higher K D for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ( Na + + K +)- ATPase can bind ouabain.

Two receptor forms for ouabain in sarcolemma-enriched preparations from canine ventricle.

Some evidence indicates that the inotropic effect of cardiac glycosides occurs at concentrations too low to affect Na+,K+-ATPase activity. This suggests that some receptor other than Na+,K+-ATPase mediates the inotropic effect. We studied ouabain binding to sarcolemma-enriched preparations from canine ventricle under conditions known to promote binding to Na+,K+-ATPase. Profiles for binding and dissociation were characterized by two kinetic components: (1) fast association and slow dissociation; (2) slow association and fast dissociation. Profiles in the absence of supporting ligands were consistent with a single species of receptors with slow association, fast dissociation and minimal effect on Na+,K+-ATPase activity. Binding supported by magnesium plus inorganic phosphate inhibited Na+,K+-ATPase activity by 86%. The two binding components were affected differentially by heating at 55 degrees C. It was concluded that the preparation possesses two receptors for ouabain: the Na+,K+-ATPase and a "new" receptor. The latter may be different chemically from the Na+,K+-ATPase. The more likely possibility is that the "new" receptor is the Na+,K+-ATPase in a state characterized by low catalytic activity, low affinity for ouabain, and no requirement of specific ligands for ouabain binding. Further, the data suggest an interdependence between the two forms. This leads to a mechanism which allows an inotropic effect to precede loss of Na+,K+-ATPase activity even though both result from glycoside binding to Na+,K+-ATPase. The mechanism involves an equilibrium between inactive and active forms of the Na+,K+-ATPase such that the inactive form buffers loss of the active form upon exposure to a cardiac glycoside.

Insulin stimulation of (Na+,K+)-adenosine triphosphatase-dependent 86Rb+ uptake in rat adipocytes.

Insulin stimulated the uptake of 86Rb+ (a K+ analog) in rat adipocytes and increased the steady state concentration of intracellular potassium. Half-maximal stimulation occurred at an insulin concentration of 200 pM. Both basal- and insulin-stimulated 86Rb+ transport rates depended on the concentration of external K+, external Na+, and were 90% inhibited by 10(-3) M ouabain and 10(-3) M KCN, indicating that the hormone was activating the (Na+,K+)-ATPase. Insulin had no effect on the entry of 22Na+ or exit of 86Rb+. Kinetic analysis demonstrated that insulin acted by increasing the maximum velocity, Vmax, of 86Rb+ entry. Inhibition of the rate of Rb+ uptake by ouabain was best described by a biphasic inhibition curve. Scatchard analysis of ouabain binding to intact cells indicated binding sites with multiple affinities. Only the rubidium transport sites which exhibited a high affinity for ouabain were stimulated by insulin. Stimulation required insulin binding to an intact cell surface receptor, as it was reversible by trypsinization. We conclude that the uptake of 86Rb+ by the (Na+,K+)-ATPase is an insulin-sensitive membrane transport process in the fat cell.

Affinity labeling of the digitalis receptor with p-nitrophenyltriazene-ouabain, a highly specific alkylating agent.

Three derivatives of ouabain have been synthesized which alkylate the digitalis receptor. These derivatives were formed through reductive amination of p-nitrophenyltriazene (NPT) ethylenediamine to the periodate-oxidized rhamnose moiety of ouabain. The non-covalent binding of the ouabain derivatives (NPT-ouabain, designated I, II, and III) was followed (i) by their ability to inhibit the activity of sodium- and potassium-activated ATPase ((Na+,K+)-ATPase) purified from the electric organ of Electrophorus electricus, (ii) by the binding of [3H]NPT-ouabain I to the enzyme, and (iii) by the inhibition of [3H]ouabain binding with unlabeled NPT-ouabain I. Covalent modification of the digitalis site of (Na+,K+)-ATPase occurs after long periods of time. At pH 7.5 (25 degrees C) the best alkylating derivative, NPT-ouabain I, gives maximum covalent labeling after 6 h. Only the large polypeptide chain (Mr = 93,000) of the purified enzyme is specifically labeled with [3H]NPT-ouabain I while the glycoprotein chain (Mr = 47,000) is not significantly labeled. Labeling of a microsomal fraction of the electric organ with [3H]NPT-ouabain I gave the same type of gel pattern as that observed with the purified enzyme. [3H]NPT-ouabain I was also used to label the digitalis receptor in highly purified axonal membranes and in cardiac membranes prepared from embryonic chick heart. Although the (Na+,K+)-ATPase in both types of membranes has a low affinity for ouabain, [3H]NPT-ouabain I proved to be a very efficient affinity label for the digitalis receptor. In the complex mixture of polypeptides found in these membrane preparations, only a single polypeptide chain having a Mr = 93,000 is specifically labeled by [3H]NPT-ouabain I.

Effects of chronic dietary lithium on activity and regulation of (Na +,K +)-adenosine triphosphatase in rat brain

Chronic dietary administration of lithium appeared to reduce (Na +,K +)-adenosine triphosphatase in rat brain by at least two mechanisms. Selective inhibition of the enzyme form with high affinity for ouabain occurred in hippocampus. Nonselective inhibition, manifested by decreased V max for activation by Na +. occurred in hippocampus, cortex and corpus striatum. There was no effect on Na + affinity. Because lithium administration decreased shock-elicited fighting behavior in the presence of moderate tissue lithium levels and in the absence of changes in weight or gross appearance or behavior, the effects on adenosine triphosphatase were pharmacologic rather than toxic.