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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Greater sensitivity to vanadate of rat renal relative to cardiac (Na++K+)-ATPase

Vanadate (VO₄^?3) produces a positive inotropic effect in rats and also promotes diuresis as well as natriuresis. Although the mechanism(s) of these effects is uncertain, in the kidney, VO₄^?3 may act through inhibition of (Na⁺+K⁺)-ATPase activity, whereas in the heart, other or additional mechanisms are likely. Under the assay conditions used in the present study, microsomal (Na⁺+K⁺)-ATPase activities from rat kidney cortex and medulla were inhibited to a greater extent than was left ventricular (Na⁺+K⁺)-ATPase activity over a range of VO₄^?3 concentrations. The apparent dissociation constant for left ventricular (Na⁺+K⁺)-ATPase (10.95 ± 1.26 × 10^?7M VO₄^?3) was significantly greater than that of (Na⁺+K⁺)-ATPase from the cortex (3.46±0.96×10^?7 M VO₄^?3) or the medulla (3.32±0.7×10^?7M VO₄^?3, N=6, P<.05) whereas there were no significant differences between the effects of VO₄^?3 on (Na⁺+K⁺)-ATPase from the cortex and medulla. The greater inhibition by VO₄, of (Na⁺+K⁺)-ATPase from the cortex relative to that of the left ventricle, occurred over a range of Na⁺ and K⁺ concentrations, and K⁺ enhanced the inhibition by VO₄^?3 to a greater extent for (Na⁺+K⁺)-ATPase from the cortex than the left ventricle. These results suggest that renal (Na⁺+K⁺)-ATPase is more sensitive than left ventricular (Na⁺+K⁺)-ATPase to inhibition by VO₄^?3 and would, therefore, be more likely to be modulated $\text{in}$$\text{vivo.}$     

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.     

A comparison of the ouabain-sensitive (Na+ + K+)-ATPase of normal and dystrophic skeletal muscle

An NaI-extraction procedure was modified to prepare muscle fiber segments with Mg²⁺-dependent, ouabain-sensitive (Na⁺ + K⁺)-ATPase activity. This enzyme was assayed in preparations of skeletal muscle from normal and dystrophic mice. The ouabain-sensitive (Na⁺ + K⁺)-ATPase activity of dystrophic muscle preparations was found to be significantly lower than that of control preparations.     

Kinetic studies on the (Na+ + K+)-dependent ATPase. Evidence for coexisting sites for Na+, K+ and Mg2+

Na⁺-ATPase activity of a dog kidney (Na⁺ + K⁺)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 μM ATP and 50 μM MgCl₂, but stimulated by 100 mM NaCl in the presence of 30 μM ATP and 3 mM MgCl₂. The $\text{K}{}_{0.5}$ for the effect of MgCl₂ was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na⁺-ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 μM MgCl₂. Similar thimerosal treatment reduced (Na⁺ + K⁺)-ATPase activity by half but did not appreciably affect the $\text{K}{}_{0.5}$ for activation by either Na⁺ or K⁺, although it reduced inhibition by high Na⁺ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na⁺: Na⁺-discharge sites at which Na⁺-binding can drive E₂-P back to E₁-P, thereby inhibiting Na⁺-ATPase activity, and sites activating E₂-P hydrolysis and thereby stimulating Na⁺-ATPase activity, corresponding to the K⁺-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na⁺-discharge and K⁺-acceptance sites. Mg²⁺ may stimulate Na⁺-ATPase activity by favoring E₂-P over E₁-P, through occupying intracellular sites distinct from the phosphorylation site or Na⁺-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na⁺-ATPase reaction and lessen Na⁺-inhibition of the (Na⁺ + K⁺)-ATPase reaction by increasing the efficacy of Na⁺ in activating E₂-P hydrolysis.     

(Na+,K+)-ATPase of mammalian brain: Effects of temperature on cation and ATP interactions regulating phosphatase activity

The effects of temperature on interactions between univalent cations or ATP and the p-nitrophenylphosphatase activity associated with brain (Na⁺,K⁺)-ATPase were examined. The apparent affinity for K⁺ activation under conditions favoring the moderate affinity site was temperature dependent, increasing with decreasing temperature. A comparison of univalent cations showed that the negative apparent ΔH and ΔS for cation binding increased with increasing apparent cation affinity. In contrast to the case with the moderate affinity sites, apparent affinity for the high affinity K⁺ site was independent of temperature. As temperature decreased, properties of moderate affinity site binding approached those of the high affinity site. The temperature dependence of ATP inhibition was opposite to that for K⁺ activation, with positive apparent ΔH and ΔS. The apparent ΔH and ΔS for cation binding approached those for the overall conformational change to K⁺-sensitive enzyme as cation affinity increased. These data suggest that E2, the K⁺-sensitive form of (Na⁺,K⁺)-ATPase, is stabilized by forces that require a decrease in entropy, explaining the predominant existence of E1 at physiologic temperatures. A conformational change leading to stabilization of E2 at higher temperatures can be produced by binding of univalent cations to a moderate affinity, presumably intracellular, site. This effect is counteracted by ATP. ATP also appears to alter the selectivity of this site to favor Na⁺ over K⁺ binding.