Estimation of ouabian specifically bound to dog renal (Na+ + K+)-ATPase in vivo

It is not known whether ouabain injected into the kidney in vivo is bound exclusively to the (Na⁺ + K⁺)-ATPase and whether the reduction of sodium pumping capacity is large enough to account for the reduction in sodium reabsorption. In the present study on dogs the total amount of parenchymal ouabain was therefore estimated and the specific renal binding compared to the reduction in (Na⁺ + K⁺)-ATPase activity. Ouabain, 120 nmol/kg body weight, was injected into the renal artery in vivo reducing the (Na⁺ + K⁺)-ATPase activity by 3lmost 80%. After nephrectomy, tissue ouabain could be quantified by radioimmunoassay after heating the homogenate to 70°C for 30 min; negligible amounts were detectable without heating. No correlation between ouabain binding and tissue volume, protein content, DNA content or Mg²⁺-ATPase content could be found when comparing the following four fractions of the kidney: outer cortex, inner cortex, outer medulla and papilla. For the whole kidney, mean parenchymal tissue concentration of ouabain equalled 0.58 ± 0.03 μmol/100 g wet tissue. Only 21.3 ± 1.2% of the ouabain was confined to the outer medulla corresponding to 54 ± 4 nmol giving a tissue concentration of 1.08 ± 0.05 μmol/100 g wet tissue. The renal ouabain concentrations were highly correlated to the reduction in (Na⁺ + K⁺)-ATPase activity, giving a ratio between the reduction in hydrolysis rate and bound ouabain (turnover number) of 6105 min^?1 which is close to the value of 7180 min^?1 found by in vitro Scatchard analysis. No ouabain seems to be bound to other tissue components than the (Na⁺ + K⁺)-ATPase and the present method is therefore a simple way of measuring the number of inhibited (Na⁺ + K⁺)-ATPase molecules after in vivo injection of ouabain.     

Showdomycin,a nucleotide-site-directed inhibitor of (Na+ + K+)-ATPase

Showdomycin [2-(β-d-ribofuranosyl)maleimide] is a nucleoside antibiotic containing a maleimide ring and which is structurally related to uridine. Showdomycin inhibited rat brain (Na⁺ + K⁺)-ATPase irreversibly by an apparently bimolecular reaction with a rate constant of about 11.01·mol^?1·min^?1. Micromolar concentrations of ATP protected against this inhibition but uridine triphosphate or uridine were much less effective. In the presence of K⁺, 100 μM ATP was unable to protect against inhibition by showdomycin. These observations show that showdomycin inhibits (Na⁺ + K⁺)-ATPase by reacting with a specific chemical group or groups at the nucleotide-binding site on this enzyme. Inhibition by showdomycin appears to be more selective for this site than that due to tetrathionate or N-ethylmaleimide. Since tetrathionate is a specific reactant for sulfhydryl groups it appears likely that the reactive groups are sulfhydryl groups. The data thus show that showdomycin is a relatively selective nucleotide-site-directed inhibitor of (Na⁺ + K⁺)-ATPase and inhibition is likely due to the reaction of showdomycin with sulfhydryl group(s) at the nucleotide-binding site on this enzyme.     

Improved purification and partial characterization of (Na+, K+)-ATPase from cardiac muscle

A method is described for purification of (N⁺, K⁺)-ATPase which yields approximately 60 mg of enzyme from 800 g of cardiac muscle with specific activities ranging from 340 to 400 μmol inorganic phosphate/mg protein per h (units/mg). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of a major 94 000 dalton polypeptide and four or five lesser components, one of which was a glycoprotein with an apparent molecular weight of 58 000. The enzyme preparation bound 600–700 pmol of [³H]ouabain/mg protein when incubated in the presence of either Mg²⁺ plus P_i or Mg²⁺ plus ATP plus Na⁺, and incorporated more than 600 pmol ³²P/mg protein when incubated with γ-³²P-labeled ATP in the presence of Mg²⁺ and Na⁺. The preparation is approximately 35% pure.     

Solvent effects on ouabain binding to the (Na+,K+)-ATPase of rat brain

The effects of the solvents deuterated water (²H₂O) and dimethyl sulfoxide (Me₂SO) on [³H]ouabain binding to (Na⁺,K⁺)-ATPase under different ligand conditions were examined. These solvents inhibited the type I ouabain binding to the enzyme (i.e., in the presence of Mg²⁺+ATP+Na⁺). In contrast, both solvents stimulated type II (i.e., Mg²⁺+P_i-, or Mn²⁺-dependent) binding of the drug. The solvent effects were not due to pH changes in the reaction. However, pH did influence ouabain binding in a differential manner, depending on the ligands present. For example, changes in pH from 7.05 to 7.86 caused a drop in the rate of binding by about 15% in the presence of Mg²⁺+Na⁺+ATP, 75% in the Mg²⁺+P_i system, and in the presence of Mn²⁺ an increase by 24% under similar conditions. Inhibitory or stimulatory effects of solvents were modified as various ligands, and their order of addition, were altered. Thus, ²H₂O inhibition of type I ouabain binding was dependent on Na⁺ concentration in the reaction and was reduced as Na⁺ was elevated. Contact of the enzyme with Me₂SO, prior to ligands for type I binding, resulted in a greater inhibition of ouabain binding than that when enzyme was exposed to Na⁺+ATP first and then to Me₂SO. Likewise, the stimulation of type II binding was greater when appropriate ligands acted on enzyme prior to addition of the solvent. Since Me₂SO and ²H₂O inhibit type I ouabain binding, it is proposed that this reaction is favored under conditions which promote loss of H₂O, and E₁ enzyme conformation; the stimulation of type II ouabain binding in the presence of the solvents suggests that this type of binding is favored under conditions which promote the presence of H₂O at the active enzyme center and E₂ enzyme conformation. This postulation of a role of H₂O in modulating enzyme conformations and ouabain interaction with them is in concordance with previous observations.     

Pressure effects on lipid-protein interactions in (Na+ + K+)-ATPase

The (Na⁺ + K⁺)-stimulated ATPase activity decreases with increasing pressure and a plot of the logarithm of the activity versus pressure shows a change in slope at a defined breakpoint pressure (P_b). The value of P_b increases linearly with increasing temperature. A $\text{d}\text{T}\text{d}\text{P}$ value of 27.7 ± 0.4 (S.D.) K/1000 atm is obtained. This is in very good agreement with the pressure shift for the melting transitions in phospholipids and aliphatic chains. This strongly indicates that an aliphatic chain melting process is involved in the breakpoint in the Arrhenius plot and pressure dependence of (Na⁺ + K⁺)-ATPase. The p-nitrophenyl phosphatase activity of this enzyme also decreases with pressure. In this case the plot of the logarithm of the activity versus pressure is linear without a break-point. The temperature dependence for (Na⁺ + K⁺)-ATPase was also studied in the presence of fluidizing drugs: desipramine and benzylalcohol. The presence of these drugs had no effect on the inflection point in the Arrhenius plot.     

Lack of stimulation of renal (Na+ + K+)-ATPase by thyroid hormones in the rabbit

The effect of l-3,5,3′-triiodothyronine (T₃) and thyroxine (T₄) on (Na⁺ + K⁺)-ATPase activities was examined in rabbit kidneys because in this tissue almost 80% of the metabolism is connected to active sodium transport. T₃-receptor concentrations were estimated as 0.62 and 0.80 pmol/mg per DNA in the cortex and outer medulla, respectively. A dose of 0.5 mg T₃/kg body weight for 3 days increased basal metabolic rate by almost 60%, and the mitochondrial 1-α-glycerophosphate dehydrogenase activity was increased by 50% in both the cortex and medulla. (Na⁺ + K⁺)-ATPase activity in the liver was raised by almost 50%. However, no changes in (Na⁺ + K⁺)-ATPase activities or binding sites for [³H]ouabain in either the kidney cortex or medulla could be observed. T₄ at 16 mg/kg daily for 14 days was also without effect on renal (Na⁺ + K⁺)-ATPase activities. Furthermore, the response to T₃ was absent at high sodium excretion rates induced by unilateral nephrectomy and extracellular volume expansion. Thus, despite stimulation of basal metabolic rate and renal 1-α-glycerophosphate dehydrogenase activity by T₃ and T₄, the (Na⁺ + K⁺)-ATPase activity in the rabbit kidney is identical in euthyroid and hyperthyroid states. However, thyroid hormones prevent the normal natriuretic response to extracellular volume expansion.     

Mechanisms of activation of renal (Na+ + K+)-ATPase in the rat Effects of acute and chronic administration of dexamethasone

The mechanisms of activation of renal (Na⁺ + K⁺)-ATPase by administration of the synthetic glucocorticoid hormone, dexamethasone, have been investigated in adrenalectomized rats. Chronic treatment with dexamethasone (1–5 mg/100 g body wt. daily for 5 days) stimulated (Na⁺ + K⁺)-ATPase specific activity in crude homogenated and microsomal fractions of renal cortex (by approx. 100–150%) and renal medulla (by approx. 100%). Acute treatment with dexamethasone (0.5–10 mg/100 g body wt.) also stimulated enzyme activity in crude homogenates and microsomal fractions of renal cortex and medulla (by approx. 40–50%). Stimulation was dose dependent and occurred within 2h after hormone treatment. In vitro addition of dexamethasone (10^?4–10^?8 M) to microsomal fractions did not modify the specific activity of (Na⁺ + K⁺)-ATPase. Stimulation of (Na⁺ + K⁺)-ATPase activity by acute and chronic administration of the hormone was demonstrated whether specific activities were expressed as a function of cellular protein or cellular DNA. Dexamethasone treatment increased the ratios protein:DNA and, to a lesser extent, the ratios RNA:DNA. However, these effects were mainly due to a reduction in the renal contents of DNA, which suggests that the observed enzyme activation is not due to an action of the hormone on renal hypertrophy. Dexamethasone also reduced cellular DNA contents in the liver. The characteristics of the activation process were essentially similar after treatment with single or multiple doses of the hormone. There were increases in the value for Na⁺ (approx. 100%), K⁺ (approx. 40%) and ATP (approx. 160%). The K_m values for Na⁺ (approx. 17 mM) and K⁺ (approx. 1.8 mM) were unchanged and there was a small increase in the K_m value for ATP (0.7 mM as against 1.7 mM). There was no difference in the Hill coefficients for the three substrates. The levels of the high-energy P_i intermediate of the (Na⁺ + K⁺)-ATPase reaction were augmented by dexamethasone treatment and the increased levels were quantitatively correlated with the observed stimulation of (Na⁺ + K⁺)-ATPase specific activity. The apparent turnover numbers of the reaction remained unchanged. The specific activity of the ouabain-sensitive p-nitrophenylphosphatase increased proportionally to the increase in (Na⁺ + K⁺)-ATPase specific activity. Enzyme activation by acute dexamethasone treatment occurred in the absence of changes in glomerular filtration rate and tubular Na⁺ excretion.These results indicate that (Na⁺ + K⁺)-ATPase activation by acute and chronic dexamethasone treatment represents an increase in the number of enzyme units with little or no change in the kinetic properties (affinity, cooperativity) of the enzyme. In addition, the information presented suggests a direct regulatory effect of glucocorticoid hormones on the activity of renal (Na⁺ + K⁺)-ATPase and is inconsistent with the concept that changes in Na⁺ loads mediate the effects of these hormones on enzyme activity. Instead, the results suggests a primary role for glucocorticoid hormones in the renal regulation of Na⁺ homeostasis.     

Potassium-p-nitrophenyl phosphate interactions with (Na+ + K+)-ATPase their relevance to phosphatase activity

The K⁺-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na⁺ + K⁺)-ATPase from pig kidney shows substrate inhibition (K_i about 9.5 mM at 2.1 mM Mg²⁺). Potassium antagonizes and sodium favours this inhibition. In addition, K⁺ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K⁺ activation. In the absence of Mg²⁺, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb⁺ from the Rb⁺ occluded unphosphorylated enzyme. With no Mg²⁺ and with 0.5 mM KCl, trypsin inactivation of (Na⁺ + K⁺)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl₂, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E₁ enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E₁ state is the basis for substrate inhibition of the p-nitrophenulphosphatase acitivy of (Na⁺ + K⁺)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.     

Exchange between inorganic phosphate and adenosine triphosphate in (Na+,K+)-ATPase

(Na⁺,K⁺)-ATPase is able to catalyze a continuous ATP?P_i exchange in the presence of Na⁺ and in the absence of a transmembrane ionic gradient. At pH 7.6 the Na⁺ concentration required for half-maximal activity is 85 mM and at pH 5.1 it is 340 mM. In the presence of optimal Na⁺ concentration, the rate of exchange is maximal at pH 6.0 and varies with ADP and P_i concentration in the assay medium. ATP?P_i exchange is inhibited by K⁺ and by ouabain.     

Substituting manganese for magnesium alters certain reaction properties of the (Na+ + K+)-ATPase

MnCl₂ was partially effective as a substitute for MgCl₂ in activating the K⁺-dependent phosphatase reaction catalyzed by a purified (Na⁺ + K⁺)-ATPase enzyme preparation from canine kidney medulla, the maximal velocity attainable being one-fourth that with MgCl₂. Estimates of the concentration of free Mn²⁺ available when the reaction was half-maximally stimulated lie in the range of the single high-affinity divalent cation site previously identified (Grisham, C.M. and Mildvan, A.S. (1974) J. Biol. Chem. 249, 3187–3197). MnCl₂ competed with MgCl₂ as activator of the phosphatase reaction, again consistent with action through a single site. However, with MnCl₂ appreciable ouabaininhibitable phosphatase activity occurred in the absence of added KCl, and the apparent affinities for K⁺ as activator of the reaction and for Na⁺ as inhibitor were both decreased. For the (Na⁺ + K⁺)-ATPase reaction substituting MnCl₂ for MgCl₂ was also partially effective, but no stimulation in the absence of added KCl, in either the absence or presence of NaCl, was detectable. Moreover, the apparent affinity for K⁺ was increased by the substitution, although that for Na⁺ was decreased as in the phosphatase reaction. Substituting MnCl₂ also altered the sensitivity to inhibitors. For both reactions the inhibition by ouabain and by vanadate was increased, as was binding of [⁴⁸V]-vanadate to the enzyme; furthermore, binding in the presence of MnCl₂ was, unlike that with MgCl₂, insensitive to KCl and NaCl. Inhibition of the phosphatase reaction by ATP was decreased with 1 mM but not 10 mM KCl. Finally, inhibition of the (Na⁺ + K⁺)-ATPase reaction by Triton X-100 was increased, but that by dimethylsulfoxide decreased after such substitution.     

Effect of magnesium ions on the high-affinity binding of eosin to the (Na+ + K+)-ATPase

(1) The fluorescence of eosin Y in the presence of (Na⁺ + K⁺)-ATPase is enhanced by Mg²⁺. The enhancement by Mg²⁺ is larger than that obtained with Na⁺ (Skou, J.C. and Esmann, M. (1981) Biochim. Biophys. Acta 647, 232–240). Mg²⁺ shifts the excitation maximum from 518 to 524 nm, the emission maximum from 538 to 542 nm. Also a shoulder appears at about 490 nm on the excitation curve, as was also observed with Na⁺. (2) The Mg²⁺-dependent enhancement of fluorescence can be reversed by K⁺ as well as by ATP. In the presence of Mg²⁺ + P_i (i.e. under conditions of phosphorylation), the fluorescence enhancement can be reversed by ouabain. With Mg²⁺ and a low concentation of K⁺ (i.e. conditions for vanadate binding), the enhancement of fluorescence can be reversed by vanadate. (3) There is a low-affinity binding of eosin which increases with the Mg²⁺ concentration. This is observed as a slight increase in the fluorescence when the excitation wavelength is above 520 nm. The low-affinity binding is K⁺-, ATP-, ouabain- and vanadate-insensitive. (4) Scatchard analysis of the binding experiments suggests that there are two high-affinity eosin-binding sites per ³²P-labelling site in the presence of 5 mM Mg²⁺ both of which are ouabain-, vanadate- and ATP-sensitive. With 5 M Mg²⁺ + 0.25 P_i, the K_d values are 0.14 μM and 1.3 μM, respectively. With 5 mM Mg²⁺, 150 mM Na⁺, the K_d values are 0.45 μM and 3.2 μM, respectively. With 5 mM Mg²⁺, the addition of K⁺ gives a pronounced decrease in affinity but does not decrease the number of binding sites (which remains at two per ³²P-labelling site). With 5 mM Mg²⁺ + 150 mM K⁺, the affinities of the two binding sites become identical, at a K_d of 17 μM. (5) The rate of conformational transitions was measured using the stopped-flow method. The rate of the transition from the Mg²⁺-form to the K⁺-form is high. Oligomycin has only a small (if any) effect on the rate. Addition of Na⁺ in the presence of Mg²⁺ does not appreciably change the rate of conversion to the K⁺-form, giving a rate constant of about 110 s^?. However, the addition of oligomycin in the presence of Mg²⁺ + Na⁺ had a profound effect: the rate of conversion to the K⁺-form was decreased by a factor of 2000 to about 0.063 s^?1. This suggests that the conformation with Mg²⁺ alone is different from the conformation with Na⁺ alone. (6) The effects of K⁺, ouabain, vanadate and ATP on the high-affinity binding of eosin suggest that the two eosin molecules bound per ³²P-labelling site are bound to ATP sites.     

The effects of long-chain alcohols on membrane lipids and the (Na+ + K+)-ATPase

The (Na⁺ + K⁺)-ATPase obtained from sheep kidney outer medulla is irreversibly denatured by long-chain aliphatic alcohols. The denaturation proceeds by causing a change in the structure of the membrane lipids rather than by binding directly to the protein. The alcohols decrease the ability of the membrane lipid bilayer to orient the spin label 3-(4′,4′-dimethyloxazolidinyl)-5α-androstan-17β-ol. For the low molecular weight alcohols the ability of the membrane to orient the label is completely lost while for alcohols with more than five carbons only partial loss of the orienting ability of the membrane occurs. The alcohol concentrations necessary to denature the enzyme correspond to the concentrations that produce the maximal change in the ability of the membrane to orient the label, and correlate well with the hydrophobicity of the alcohols as measured by their water-octanol partition coefficients.     

Ouabain binding sites and the (Na+,K+)-ATPase of brain microsomal membranes

Beef brain microsomes bound approximately 180–220 pmoles of [³H]ouabain per mg of protein in the presence of either MgCl₂ and inorganic phosphate or ATP, MgCl₂ and NaCl. The ouabain-binding capacity and the ouabain-membrane complex were more stable than the (Na⁺,K⁺)-ATPase activity to treatment with agents known to affect the membrane integrity, such as, NaClO₄, sodium dodecyl sulfate, $\text{p-}\text{chloromercuribenzoate}$, urea. ultrasonication, heating, pH and phospholinase C.The presence of binding sites that were normally inaccessible to ouabain in brain microsomes was demonstrated. These sites appeared after disruption of microsomes with 2 M NaClO₄ as evidenced by increased binding of [³H]ouabain. These sites may be buried during the subcellular fractionation procedure and could be accessible in the intact cell.     

Studies on the interaction of chick brain microsomal (Na+ + K+)-ATPase with copper

Chick brain microsomal ATPase was strongly inhibited by Cu²⁺. (Na⁺ + K⁺)-ATPase was more susceptible to low levels of Cu²⁺ than Mg²⁺-ATPase. The inhibition of (Na⁺ + K⁺)-ATPase could be partially protected from Cu²⁺ in the presence of ATP in the preincubation period. When Cu²⁺ (6 μM) was preincubated with the enzyme in the absence of ATP, only sulfhydryl-containing amino acids (d-penicillamine and l-cysteine) could reverse the inhibition. At lower concentrations of Cu²⁺ (< 1.4 μM), in the absence of ATP during preincubation, the inhibition could be completely reversed by the addition of 5 mM l-phenylalanine and l-histidine as well as d-penicillamine and l-cysteine.Kinetic analysis of action of Cu²⁺ (1.0 μM) on (Na⁺ + K⁺)-ATPase revealed that the inhibition was uncompetitive with respect to ATP. At a low concentration of K⁺ (5 mM), V with Na⁺ was markedly decreased in the presence of Cu²⁺ and K_m was about twice that of the control. However, at high K⁺ concentration (20 mM), the K_m for Na⁺ was not affected. At both low (25 mM) and high (100 mM) Na⁺, Cu²⁺ displayed non-competitive inhibition of the enzyme with respect to K⁺.On the basis of these data, we suggest that Cu²⁺ at higher concentrations (> 6 μM) inactivates the enzyme irreversibly, but that at lower concentrations (< 1.4 μM), Cu²⁺ interacts reversibly with the enzyme.     

Mechanism of the involvement of monoamine oxidase in the regulation of (Na+ + K+)-ATPase in rat brain

Increasing concentrations of dopamine fail to give a biphasic response to (Na⁺ + K⁺)-ATPase activity in various subcellular fractions of rat brain preincubated with monoamine oxidase inhibitors, viz. 1·10^?4 M clorgyline and 1·10^?4 M deprenyl. The product of the monoamine-oxidase-catalysed reaction with dopamine as substrate is 3-methoxy-4-hydroxyphenylacetaldehyde. An analogue of this product is 3-methoxy-4-hydroxybenzaldehyde. This analogue, when incubated with the subcellular fractions which had been preincubated with monoamine oxidase inhibitors and dopamine, gave a more pronounced biphasic response to (Na⁺ + K⁺)-ATPase activity than that observed in the fractions incubated with dopamine alone.     

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.     

Temperature effects on cation affinities of the (Na+, K+)-ATPase of mammalian brain

Effects of temperature on the Na⁺-dependent ADP-ATP exchange and the $\text{p-}\text{nitrophenylphosphatase}$ reactions catalysed by (Na⁺, K⁺)-ATPase were examined. Apparent Mg²⁺ affinity decreased with decreasing temperature. Arrhenius plots of $\text{p-}\text{nitrophenylphosphatase}$ in the presence of Na⁺ and ATP had discontinuities similar to those previously reported for ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$, while those of $\text{p-}\text{nitrophenylphosphatase}$ measured without Na⁺ or ATP did not. The apparent activation energy for $\text{p-}\text{nitrophenylphosphatase}$ was a function of the physical characteristics of the cation acting at the K⁺ site.