Two gears of pumping by the sodium pump

The kinetics of the phosphorylation and subsequent conformational change of Na⁺,K⁺-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24°C). The enzyme was preequilibrated in buffer containing 130 mM NaCl to stabilize the E1(Na⁺)₃ state. On mixing with ATP in the presence of Mg²⁺, a fluorescence increase occurred, due to enzyme conversion into the E2P state. The fluorescence change accelerated with increasing ATP concentration until a saturating limit in the hundreds of micromolar range. The amplitude of the fluorescence change (ΔF/F₀) increased to 0.98 at 50 μM ATP. ΔF/F₀ then decreased to 0.82 at 500 μM. The decrease was attributed to an ATP-induced allosteric acceleration of the dephosphorylation reaction. The ATP concentration dependence of the time course and the amplitude of the fluorescence change could not be explained by either a one-site monomeric enzyme model or by a two-pool model. All of the data could be explained by an (αβ)₂ dimeric model, in which the enzyme cycles at a low rate with ATP hydrolysis by one α-subunit or at a high rate with ATP hydrolysis by both α-subunits. Thus, we propose a two-gear bicyclic model to replace the classical monomeric Albers-Post model for kidney Na⁺,K⁺-ATPase.     

The mode of activation by potassium of potassium-dependent and ouabain-sensitive phosphatase activity.

A new simple procedure has been developed for the purification of plasma membranes from rabbit kidney microsomes which yields a three- to fourfold increase in the specific activity of Na⁺-K⁺-adenosine triphosphatase (ATPase). The procedure differs from previous methods with deoxycholate or other detergents and does not change the molecular activity of the ATPase. The K⁺-dependent p-nitrophenylphosphatase activity of the native Na⁺-K⁺-ATPase is controlled more effectively by Mg²⁺ in the presence of K⁺ at concentrations higher than that of Mg²⁺, and by K⁺ in the presence of Mg²⁺ at concentrations higher than that of K⁺. The enzyme in its Mg²⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 1, is less sensitive to ouabain (I_0.5 = 90 μM) and corresponds to the enzyme conformation reported previously which is inhibited by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin (I_0.5 is the midpoint of the saturation curve). The enzyme in its K⁺-regulating state, which shows K⁺-saturation curves with a Hill coefficient of 2, is more sensitive to ouabain inhibition (I₀₅ = 8 μM) and corresponds to the enzyme conformation which is stimulated by the concurrent presence of Na⁺ and ATP or of Na⁺ and oligomycin. There appear to be two conformations of the enzyme that are regulated by Mg²⁺ binding on the inhibitory sites of the enzyme.     

Dual Mechanisms of Allosteric Acceleration of the Na,K-ATPase by ATP

Investigations of the E2 → E1 conformational change of Na⁺,K⁺-ATPase from shark rectal gland and pig kidney via the stopped-flow technique have revealed major differences in the kinetics and mechanisms of the two enzymes. Mammalian kidney Na⁺,K⁺-ATPase appears to exist in a diprotomeric (αβ)₂ state in the absence of ATP, with protein-protein interactions between the α-subunits causing an inhibition of the transition, which occurs as a two-step process: E2:E2 → E2:E1 → E1:E1. This is evidenced by a biphasicity in the observed kinetics. Binding of ATP to the E1 or E2 states causes the kinetics to become monophasic and accelerate, which can be explained by an ATP-induced dissociation of the diprotomer into separate αβ protomers and relief of the preexisting inhibition. In the case of enzyme from shark rectal gland, the observed kinetics are monophasic at all ATP concentrations, indicating a monoprotomeric enzyme; however, an acceleration of the E2 → E1 transition by ATP still occurs, to a maximum rate constant of 182 (± 6) s⁻¹. This indicates that ATP has two separate mechanisms whereby it accelerates the E2 → E1 transition of Na⁺,K⁺-ATPase αβ protomers and (αβ)₂ diprotomers.     

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.     

Structural Changes in the Catalytic Cycle of the Na,K-ATPase Studied by Infrared Spectroscopy

Pig kidney Na⁺,K⁺-ATPase was studied by means of reaction-induced infrared difference spectroscopy. The reaction from E1Na₃⁺ to an E2P state was initiated by photolysis of P³-1-(2-nitrophenyl)ethyl ATP (NPE caged ATP) in samples that contained 3 mM free Mg²⁺ and 130 mM NaCl at pH 7.5. Release of ATP from caged ATP produced highly detailed infrared difference spectra indicating structural changes of the Na⁺,K⁺-ATPase. The observed transient state of the enzyme accumulated within seconds after ATP release and decayed on a timescale of minutes at 15°C. Several controls ensured that the observed difference signals were due to structural changes of the Na⁺,K⁺-ATPase. Samples that additionally contained 20 mM KCl showed similar spectra but less intense difference bands. The absorbance changes observed in the amide I region, reflecting conformational changes of the protein backbone, corresponded to only 0.3% of the maximum absorbance. Thus the net change of secondary structure was concluded to be very small, which is in line with movement of rigid protein segments during the catalytic cycle. Despite their small amplitude, the amide I signals unambiguously reveal the involvement of several secondary structure elements in the conformational change. Similarities and dissimilarities to corresponding spectra of the Ca²⁺-ATPase and H⁺,K⁺-ATPase are discussed, and suggest characteristic bands for the E1 and E2 conformations at 1641 and 1661 cm⁻¹, respectively, for αβ heterodimeric ATPases. The spectra further indicate the participation of protonated carboxyl groups or lipid carbonyl groups in the reaction from E1Na₃⁺ to an E2P state. A negative band at 1730 cm⁻¹ is in line with the presence of a protonated Asp or Glu residue that coordinates Na⁺ in E1Na₃⁺. Infrared signals were also detected in the absorption regions of ionized carboxyl groups.     

Studies on (NA + K)-activated ATPase XLV. Magnesium induces two low-affinity non-phosphorylating nucleotide binding sites per molecule

ATP and adenylylimidodiphosphate (AdoPP[NH]P) bind to (Na⁺ + K⁺)-ATPase in the absence of Mg²⁺ (EDTA present) with a homogeneous but 15-fold different affinity, the K_d values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na⁺ + K⁺)-ATPase, respectively. The K_d value for ATP is equal to the K_m for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na⁺ + K⁺)-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg²⁺, as shown by binding studies with the non-phosphorylating (AdoPP[NH]P). The K_d and binding capacity for AdoPP[NH]P at these sites is dependent on the Mg²⁺ concentration. The K_d increases from 0.06 mM at 0.5 mM Mg²⁺ to a maximum of 0.26 mM at 2 mM Mg²⁺ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg²⁺ to 3.3 nmol/mg protein at 4 mM Mg²⁺. Extrapolation of a double reciprocal plot of binding capacity vs. total Mg²⁺ concentration yields a maximal binding capacity at infinite Mg²⁺ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na⁺ + K⁺)-ATPase. The K_d for Mg²⁺ at the sites, where it exerts this effect, is 0.8 mM. The K_d for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg²⁺ to a maximum of 4.2 μM at 2 mM Mg²⁺ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg²⁺ concentration. Hence, Mg²⁺ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na⁺ + K⁺)-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites.     

Properties and nature of (Na+ + Mg2+)-dependent adenosine triphosphatase in the gills of the eel, angvilla anguilla (L.)

The specific activity of (Na⁺ + Mg²⁺)-dependent ATPase is three times greater in the microsomes of sea-water eels than in freshwater eels; the specific activity is one quarter of that of (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase in both cases.(Na⁺ + Mg²⁺)-dependent ATPase is optimally active in a medium containing 8 mM NaCl, 4 mM MgCI₂, 4 mM ATP, pH 8.8 and at 30 °C; the enzyme is inhibited by ouabain, by NaCl concentrations > 100 mM and by treatment with urea.It is concluded that the (Na⁺ + Mg²⁺)-dependent ATPase activity of gills arises from the presence of a (Na⁺ + K⁺ + Mg²⁺)-dependent ATPase.     

Alterations in phospholipid-dependent (Na++K+)-ATPase activity due to lipid fluidity: Effects of cholesterol and Mg2+

The (Na⁺+K⁺)-activated, Mg²⁺-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble from depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na⁺+K⁺)-ATPase in its pH optimum being around 7.0 showing optimal activity at Mg²⁺: ATP mol ratios of approximately 1 and a K_m value for ATP of 0.4 mM.Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg²⁺: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 °C, With activation energy (E_a) values of 13–15 kcal/mol above this temperature and 30–35 kcal below it. A further discontinuity was also found at 8.0 °C and the E_a below this was very high (> 100 kcal/mol).Incresed Mg²⁺ concentrations at Mg²⁺: ATP ratios in excess of 1:1 inhibited the (Na⁺+K⁺)-ATPase activity and also abolished the discontinuities in the Arrhenius plots.The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na⁺+K⁺)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20°C and E_a values of 22 and 68kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg²⁺-ATPase normally showed a linear Arrhenius plot with an E_a of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg²⁺-ATPase activity, which now also showed a discontinuity at 20 °C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the K_m for ATP.Since both of cholesterol and Mg²⁺ are know to alter the effects of temperature on the fluidity of phospholipids the above result are discussed in this context.     

Properties of the Mg2+-induced low-affinity nucleotide binding site of (Na++ K+)-activated ATPase

(1) The Mg²⁺-induced low-affinity nucleotide binding by (Na⁺ + K⁺)-ATPase has been further investigated. Both heat treatment (50–65°C) and treatment with N-ethylmaleimide reduce the binding capacity irreversibly without altering the K_d value. The rate constant of inactivation is about one-third of that for the high-affinity site and for the (Na⁺ + K⁺)-ATPase activity. (2) Thermodynamic parameters (ΔH° and ΔS°) for the apparent affinity in the ATPase reaction (K_m ATP) and for the true affinity in the binding of AdoPP[NH]P (K_d and K_i) differ greatly in sign and magnitude, indicating that one or more reaction steps following binding significantly contribute to the K_m value, which thus is smaller than the K_d value. (3) Ouabain does not affect the capacity of low-affinity nucleotide binding, but only increases the K_d value to an extent depending on the nucleotide used. GTP and CTP appear to be most sensitive, ATP and ADP intermediately sensitive and AdoPP[NH]P and least sensitive to ouabain. Ouabain reduces the high-affinity nucleotide binding capacity without affecting the K_d value. (4) The nucleotide specificity of low-affinity binding site is the same for binding (competition with AdoPP[NH]P) and for the ATPase activity (competition with ATP): AdoPP[NH]P > ATP > ADP > AMP. (5) The low-affinity nucleotide binding capacity is preserved in the ouabain-stabilized phosphorylated state, and the K_d value is not increased more than by ouabain alone. (6) It is inferred that the low-affinity site is Iocated on the enzyme, more specifically its α-subunit, and not on the surrounding phospholipids. It is situated outside the phosphorylation centre. The possible functional role of the low-affinity binding is discussed.     

Differences between CTP and ATP as substrates for the (Na + K)-ATPase

CTP was a poorer substrate than ATP when substituted in the (Na + K)-ATPase reaction assay, not only in terms of K_m but also of V. CDP was a poorer inhibitor than ADP, so product inhibition cannot account for CTP being a poorer substrate. In the Na-ATPase reaction, which the enzyme also catalyzes, substituting CTP for ATP resulted in greater activity, arguing against CTP being less effective than ATP in forming the enzyme-phosphate intermediate common to both reactions. Ligands that favor the E₂ conformational state of the enzyme, K⁺, Mg²⁺, and Mn₂⁺, inhibited the (Na + K)-CTPase reaction more than the (Na + K)-ATPase. Conversely, Triton X-100, which favors the E₁ conformational state of the enzyme, K⁺, Mg²⁺, and Mn²⁺, inhibited the (Na + K)-CTPase ATPase reaction but stimulated the (Na + K)-CTPase. Although the (Na + K)-ATPase reaction sequence probably involves cyclical interconversion between E₁ and E₂ conformational states (and is thus inhibitable by ligands favoring either state), the K-phosphatase reaction catalyzed by the enzyme apparently functions entirely in the E₂ state. This reaction is better stimulated by CTP plus Na⁺ than by ATP plus Na⁺; moreover, CTP lessens inhibition by Triton X-100, and ATP lessens inhibition by inorganic phosphate (which reacts with the E₂ state). These observations indicate that CTP is a poorer substrate than ATP because it is less effective in promoting conversion of E₂ to E₁, essential for the (Na + K)-dependent reaction mechanism. However, contrary to this rationale, dimethyl sulfoxide stimulated the (Na + K)-CTPase reaction although by other criteria, including inhibition of the (Na + K)-ATPase, the reagent appears to favor the E₂ over the E₁ conformational state.     

Erythrocyte Membrane Digoxin-Sensitive (Na+–K+)-ATPase of Non-Insulin Dependent Diabetic Humans

Erythrocyte plasma membranes of non-insulin dependent diabetic humans (NIDDM) and healthy humans were prepared by hypotonic lysis. The specific activity of (Na⁺–K⁺)-ATPase of NIDDM membranes, both in the absence and presence of digoxin were lower than the specific activity of normal enzymes (83.6 percent and 74.0 percent of the normal enzyme respectively). Addition of digoxin decreased the activity of this enzyme (38.0 percent in NIDDM and 30.0 percent in normal enzyme).Although the affinity of the pump for ATP was similar in both membranes of NIDDM and normal humans (K_m for ATP=19.9±0.24M ATP and 20.0±0.21 M ATP respectively), the V_max of NIDDM membranes was more than 20 percent lower than that of the normal enzyme. The specific activity of Mg²⁺-dependent Ca²⁺-pumping ATPase (Ca²⁺–Mg²⁺)-ATPase) of NIDDM membrane was lower than 80 percent of the specific activity of the normal enzymes. While the affinity of the pump for ATP was lower in the membranes of NIDDM (K_m for ATP=50.0±4.3 M ATP) in comparison to normal membranes (K_m for ATP=63.1±38M ATP), the V_max of NIDDM membranes was similar to the normal enzyme. Altogether, these findings suggest that both the (Na⁺–K⁺)-ATPase and Ca²⁺-pumping ATPase of NIDDM membranes are less functional than the enzymes in normal erythrocytes.     

Characterization of microsomal ATPases from developing human placenta

Activities and some properties of microsomal ATPases have been studied in developing human placenta. The enzyme activities (Na⁺ + K⁺ + Mg²⁺, Mg²⁺, and Ca²⁺ dependent) in the placenta increase steadily with gestational age until the 18th to 21st week, and decrease in the second half of pregnancy. Mg²⁺-dependent and Na⁺ + K⁺ + Mg²⁺-dependent ATPases possess nearly the same K_m (apparent) for ATP, while the Ca²⁺-dependent enzyme shows a different one. Mg²⁺-dependent ATPase shows higher substrate affinity than Ca²⁺-dependent ATPase, although the V_max of the Mg²⁺-dependent enzyme is lower than that of the latter. However, for each enzyme, the K_m remains almost constant and V_max varies during ontogenic development. V_max of the enzymes decline at term. The enzymes are heat-labile, unaffected by amino acids, namely, l-phenylalanine, l-leucine, and l-tryptophan, and deoxycholate inhibits the enzyme activities by about 50%.     

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.     

ATP and magnesium drive conformational changes of the Na/K-ATPase cytoplasmic headpiece

Conformational changes of the Na⁺/K⁺-ATPase isolated large cytoplasmic segment connecting transmembrane helices M4 and M5 (C45) induced by the interaction with enzyme ligands (i.e. Mg²⁺ and/or ATP) were investigated by means of the intrinsic tryptophan fluorescence measurement and molecular dynamic simulations. Our data revealed that this model system consisting of only two domains retained the ability to adopt open or closed conformation, i.e. behavior, which is expected from the crystal structures of relative Ca²⁺-ATPase from sarco(endo)plasmic reticulum for the corresponding part of the entire enzyme. Our data revealed that the C45 is found in the closed conformation in the absence of any ligand, in the presence of Mg²⁺ only, or in the simultaneous presence of Mg²⁺ and ATP. Binding of the ATP alone (i.e. in the absence of Mg²⁺) induced open conformation of the C45. The fact that the transmembrane part of the enzyme was absent in our experiments suggested that the observed conformational changes are consequences only of the interaction with ATP or Mg²⁺ and may not be related to the transported cations binding/release, as generally believed. Our data are consistent with the model, where ATP binding to the low-affinity site induces conformational change of the cytoplasmic part of the enzyme, traditionally attributed to E2 → E1 transition, and subsequent Mg²⁺ binding to the enzyme-ATP complex induces in turn conformational change traditionally attributed to E1 → E2 transition.     

Hydrolysis of adenylyl imidodiphosphate in the presence of Na+ + Mg+ by (Na+ + K+)-activated ATPase

(1) Contrary to what has usually been assumed, (Na⁺ + K⁺)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na⁺ + Mg²⁺ to ADP-NH₂ and P_i. The activity is ouabain-sensitive and is not detected in the absence of either Mg²⁺ or Na²⁺. The specific activity of the Na⁺ + Mg²⁺ dependent AdoPP[NH]P hydrolysis at 37°C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K⁺. The activity is not stimulated by K⁺, nor can K⁺ replace Na⁺ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na⁺ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5–9 mM. The K_m value for AdoPP[NH]P is 17 μM. At 0°C and 21°C the specific activity is 2 and 14%, respectively, of that at 37°C. AMP, ADP and AdoPP[CH₂]P are not detectably hydrolysed by (Na⁺ + K⁺)-ATPase in the presence of Na⁺ + Mg²⁺. (2) In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37°C of 0.0006, 0.006 and 0.07 h^?1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg²⁺ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h^?1 at 37°C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. (3) A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na⁺ + Mg²⁺ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0°C. On the other hand, determination of affinity in the (Na⁺ + K⁺)-ATPase reaction by competition with ATP at low protein concentrations (μg/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37°C.     

Fluorescent labeling of (Na + K)-ATPase by 5-iodoacetamidofluorescein

5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na⁺ + K⁺)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K⁺-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na⁺, K⁺, Mg²⁺, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na⁺, and Mg²⁺, and decreased by K⁺. These fluorescence changes likely reflect ligand-induced stabilization of the E₁ or E₂ states of the enzyme.     

Conformational changes of membrane-bound (Na+−K+)-ATPase as revealed by antibody inhibition

Summary As different structural states of the (Na⁺–K⁺)-ATPase (EC 3.6.1.3) may lead to a changed reactivity to antibodies, the influence of Na⁺, K⁺, Mg⁺⁺, P_i and ATP on the reaction between highly purified (Na⁺–K⁺)-ATPase and antibodies directed against the membrane-bound enzyme was measured. The antigen antibody reaction was registered by measuring the antibody inhibition of (Na⁺–K⁺)-ATPase activity.In themembrane-bound but not in thesolubilized enzyme four different degrees of antibody inhibition were obtained at equilibrium of the antigen antibody reaction if different combinations of Na⁺, K⁺, Mg⁺⁺ and ATP were present during the incubation with the antibodies. Corresponding to the different degrees of inhibition, different rates of enzyme inhibition were measured. (a) The smallest degree of enzyme inhibition was obtained when (i) only Mg⁺⁺, (ii) Mg⁺⁺ and Na⁺ or (iii) Mg⁺⁺ and K⁺ were present during the antigen antibody reaction. (b) The enzyme activity was inhibited more strongly if Na⁺, Mg⁺⁺ and ATP were present together. (c) It was inhibited even more if only (i) Na⁺, (ii) K⁺, (iii) ATP or both (iv) ATP and Na⁺, (v) ATP and K⁺, (vi) ATP and Mg⁺⁺, or if (vii) no ATP and activating ions were present. (d) The highest degree of antibody inhibition was obtained if Mg⁺⁺, ATP and K⁺ were present together.In the presence of Mg⁺⁺ plus ADP and in the presence of Mg⁺⁺ plus the ATP analog adenylyl (--methylene) diphosphonate, Na⁺ and K⁺ did not influence the degree of antibody inhibition as they did in the presence of Mg⁺⁺ plus ATP. It was further found that the degree of antibody inhibition in the presence of Mg⁺⁺, ATP and K⁺ was affected by the sequence in which K⁺ and ATP were added to the enzyme prior to the addition of the antibodies.It is suggested that by antibody inhibition different conformations of the (Na⁺–K⁺)-ATPase could be detected. These conformations may possibly not occur in the solubilized enzyme and therefore do not seem to be necessarily linked to the intermediary steps of the ATP hydrolysis of the enzyme. The structural changes which are induced by Na⁺ and K⁺ in the presence of Mg⁺⁺ plus ATP are proposed to occur during the Na⁺–K⁺ transport.