Modification of the (Na+ + K+)-dependent ATPase by acetic anhydride and trinitrobenzene sulfonate: Specific changes in enzymatic properties

Acetic anhydride irreversibly inactivated (Na⁺ + K⁺)-dependent ATPase preparations from brain, kidney, and eel electroplax. The extent of inactivation was dose dependent, and varied also with the pH of the medium, inactivation decreasing with pH in the range 8.4 to 6.7. Including KCl (k_0.5 ca. 0.6 mm) or ATP (K_0.5 ca. 1 μm) in the medium protected against inactivation, whereas MgCl₂ (k_0.5 ca. 1 mm) increased inactivation. K⁺-Dependent phosphatase activity of the enzyme was lost in parallel with (Na + K)-ATPase activity, but Na⁺-dependent phosphorylation of the enzyme and Na⁺-dependent ATPase activity were relatively resistant to inactivation. Extraction of the membrane lipids of treated enzyme preparations and replacement with exogenous lipid dispersions did not reverse the inactivation; on the other hand, the catalytic peptide of the enzyme was labeled after incubation with radioactive acetic anhydride. For the enzymatic activity remaining after treatment with acetic anhydride several kinetic properties were also modified. For the K-phosphatase reaction the k_0.5 for K⁺-activation was greatly increased, whereas for the (Na + K)-ATPase reaction the k_0.5 for neither K⁺ nor Na⁺ was increased, although the apparent k_m for ATP was decreased. These observations are interpreted in terms of a decreased apparent affinity for K⁺ at the moderate-affinity α sites of the enzyme, sites involved in (i) activating the K-phosphatase but not the (Na + K)-ATPase reactions and (ii) influencing the k_m for ATP. Effects of trinitrobenzene sulfonate (TNBS) on the enzyme preparations were similar: Both KCl and ATP reduced the extent of irreversible inactivation; the pH dependence indicated a pK_a for the reactive enzyme groups of 7.5–8; and TNBS affected K⁺-activation analogously. Moreover, inactivation by acetic anhydride and TNBS followed the pattern of mutually exclusive inhibitors, and prior treatment with TNBS reduced labeling of the enzyme by radioactive acetic anhydride. By contrast, partial inactivation by pyridoxal phosphate or N-ethylmaleimide did not result in a similarly modified enzyme. The effects of acetic anhydride and TNBS appear to be mediated (at least in part) through amino groups not accessible to or reactive with the other reagents: groups which influence the moderate-affinity α sites and which are protected by the presence of K⁺ at these sites.     

Vanadate binding to the (Na + K)-ATPase

A particulate (Na + K)-ATPase preparation from dog kidney bound [⁴⁸V]-ortho-vanadate rapidly at 37°C through a divalent cation-dependent process. In the presence of 3 mM MgCl₂ theK _d was 96 nM; substituting MnCl₂ decreased theK _d to 12 nM but the maximal binding remained the same, 2.8 nmol per mg protein, consistent with 1 mol vanadate per functional enzyme complex. Adding KCl in the presence of MgCl₂ increased binding, with aK _0.5 for KCl near 0.5 mM; the increased binding was associated with a drop inK _d for vanadate to 11 nM but with no change in maximal binding. Adding NaCl in the presence of MgCl₂ decreased binding markedly, with anI ₅₀ for NaCl of 7 mM. However, in the presence of MnCl₂ neither KCl nor NaCl affected vanadate binding appreciably. Both the nonhydrolyzable, ,-imido analog of ATP and nitrophenyl phosphate, a substrate for the K-phosphatase reaction that this enzyme also catalyzes, decreased vanadate binding at concentrations consistent with their acting at the low-affinity substrate site of the enzyme; the presence of KCl increased the concentration of each required to decrease vanadate binding. Oligomycin decreased vanadate binding in the presence of MgCl₂, whereas dimethyl sulfoxide and ouabain increased it. With inside-out membrane vesicles from red blood cells vanadate inhibited both the K-phosphatase and (Na + K)-ATPase reactions; however, with the K-phosphatase reaction extravesicular K⁺ (corresponding to intracellular K⁺) both stimulated catalysis and augmented vanadate inhibition, whereas with the (Na + K)-ATPase reaction intravesicular K⁺ (corresponding to extracellular K⁺) both stimulated catalysis and augmented vanadate binding.     

Effects of Cations on (Ca2++ Mg2+)-Activated ATPase from Rat Brain

Abstract: With a partially purified, membrane-bound (Ca + Mg)-activated ATPase preparation from rat brain, the K_0.5 for activation by Ca²⁺ was 0.8 p μm in the presence of 3 mm -ATP, 6 mm -MgCl₂, 100 m_M-KCI, and a calcium EGTA buffer system. Optimal ATPase activity under these circumstances was with 6-100 μm -Ca²⁺, but marked inhibition occurred at higher concentrations. Free Mg²⁺ increased ATPase activity, with an estimated K_0.5, in the presence of 100 μm -CaCl₂, of 2.5 mm ; raising the MgCl₂ concentration diminished the inhibition due to millimolar concentrations of CaCl₂, but antagonized activation by submicromolar concentrations of Ca²⁺. Dimethylsulfoxide (10%, v/v) had no effect on the K_0.5 for activation by Ca²⁺, but decreased activation by free Mg²⁺ and increased the inhibition by millimolar CaCl₂. The monovalent cations K⁺, Na⁺, and TI⁺ stimulated ATPase activity; for K⁺ the K_0.5 was 8 mm , which was increased to 15 mm in the presence of dimethylsulfoxide. KCI did not affect the apparent affinity for Ca²⁺ as either activator or inhibitor. The preparation can be phosphorylated at 0°C by [γ-³²P]-ATP; on subsequent addition of a large excess of unlabeled ATP the calcium dependent level of phosphorylation declined, with a first-order rate constant of 0.12 s^?1. Adding 10 mm -KCI with the unlabeled ATP increased the rate constant to 0.20 s^?1, whereas adding 10 mm -NaCl did not affect it measurably. On the other hand, adding dimethyl-sulfoxide slowed the rate of loss, the constant decreasing to 0.06 s^?1. Orthovanadate was a potent inhibitor of this enzyme, and inhibition with 1 μm -vanadate was increased by both KCI and dimethylsulfoxide. Properties of the enzyme are thus reminiscent of the plasma membrane (Na + K)-ATPase and the sarcoplasmic reticulum (Ca + Mg)-ATPase, most notably in the K⁺ stimulation of both dephosphorylation and inhibition by vanadate.     

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.     

Binding to the high-affinity substrate site of the (Na+ + K+)-dependent ATPase

The (Na⁺ + K⁺)-dependent ATPase exhibits substrate sites with both high affinity (K _m near 1 µM) and low affinity (K _m near 0.1 mM) for ATP. To permit the study of nucleotide binding to the high-affinity substrate sites of a canine kidney enzyme preparation in the presence as well as absence of MgCl₂, the nonhydrolyzable - imido analog of ATP, AMP-PNP, was used in experiments performed at 0–4°C by a centrifugation technique. By this method theK _D for AMP-PNP was 4.2 µM in the absence of MgCl₂. Adding 50 µM MgCl₂, however, decreased theK _D to 2.2 µM; by contrast, higher concentrations of MgCl₂ increased theK _D until, with 2 mM MgCl₂, theK _D was 6 µM. The half-maximal effect of MgCl₂ on increasing theK _D occurred at approximately 1 mM. This biphasic effect of MgCl₂ is interpreted as Mg²⁺ in low concentrations favoring AMP-PNP binding through formation at the high-affinity substrate sites of a ternary enzyme-AMP-PNP-Mg complex; inhibition of nucleotide binding at higher MgCl₂ concentrations would represent Mg²⁺ acting through the low-affinity substrate sites. NaCl in the absence of MgCl₂ increased AMP-PNP binding, with a half-maximal effect near 0.3 mM; in the presence of MgCl₂, however, NaCl increased theK _D for AMP-PNP. KCl decreased AMP-PNP binding in the presence or absence of MgCl₂, but the simultaneous presence of a molar excess of NaCl abolished (or masked) the effect of KCl. ADP and ATP acted as competitors to the binding of AMP-PNP, although a substrate for the K⁺-dependent phosphatase reaction also catalyzed by this enzyme,p-nitrophenyl phosphate, did not. This lack of competition is consistent with formulations in which the phosphatase reaction is catalyzed at the low-affinity substrate sites.     

(Ca + Mg)-stimulated ATPase activity of a rat brain microsomal preparation.

ATPase activity of freshly prepared brain microsomes was stimulated 20% when 0.1 mm CaCl₂ was added in the presence of a “saturating” concentration of MgCl₂ (4 mm). This (Ca + Mg)-stimulated activity declined rapidly on storage. Treatment of the microsomes with 0.12% deoxycholate in 0.15 m KCl, followed by centrifugation and resuspension in sucrose, produced a preparation both stable on storage at ?15 °C and with an increased stimulation in the presence of CaCl₂. SrCl₂ was more effective than CaCl₂, but BaCl₂ was a poor activator. KCl and NaCl stimulated the (Ca + Mg)-ATPase activity by reducing substrate (ATP) inhibition. The K_m for ATP was 0.1 mm, a third that of the Mg-ATPase. CTP, ITP, and GTP could not substitute for ATP, although they were fair substrates for the Mg-ATPase. The energy of activation of the (Ca + Mg)-ATPase was 21 kcal, nearly twice that of the Mg-ATPase. After sucrose density-gradient centrifugation of the microsomal preparation, the (Ca + Mg)-ATPase activity was distributed with the (Na + K)-ATPase and not with the mitochondrial marker succinic dehydrogenase. Studies with ouabain, oligomycin, and azide distinguished the (Ca + Mg)-stimulated ATPase from (Na + K)- and mitochondrial ATPases. Sensitivity to ruthenium red suggested a link to Ca transport, although the microsomal ⁴⁵Ca accumulating system was much more sensitive to the inhibitor than was this ATPase activity.     

Variable affinity of the (Na+ + K+)-dependent adenosine triphosphatase for potassium: Studies using beryllium inactivation

Inactivation of the (Na⁺ + K⁺)-dependent ATPase by 50 μm BeCl₂ occurred during brief incubations in the presence of both Mg²⁺ and K⁺. Inactivation followed, initially, a first-order time course, with rate constants sensitive to the concentration of K⁺ (other components held constant). From these data dissociation constants can be calculated for K⁺ binding to sites controlling inactivation. Comparisons of relative affinities for K⁺ analogs (T1⁺ and NH₄⁺), and of sensitivity to reagents altering K⁺ activation (phlorizin and dimethylsulfoxide) indicate that the same K⁺ sites operate for both Be²⁺ inactivation and enzyme activation. With 3 mm MgCl₂ the dissociation constant, K_D, for K⁺ was 1.4 mm, but decreased 20-fold on addition of both Na⁺ and CTP. Alone, Na⁺ increased the apparent K_D for K⁺, either by direct competition or indirectly from its own site, with a K_D of 7 mm. The data suggest a model for K⁺ transport with K⁺ sites on the outer membrane surface that increase in affinity after formation of the phosphorylated enzyme intermediate, sufficiently to bind K⁺ in a high Na⁺ environment. Translocation may occur by an “oscillating pore” mechanism discharging K⁺ at the inner surface, while leaving demonstrable sites of moderate affinity at the outer end of the pore (which preclude attempts to document low-affinity discharge sites).     

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.     

Substrate sites for the (Na+ + K+)-dependent ATPase.

Kinetic studies on a rat brain (Na+ + K+)-dependent ATPase (EC 3.6.1.3) preparation demonstrated high-affinity sites for ATP, with a Km near 1 mum, and low affinity sites for ATP, with a Km near 0.5 mM. In addition, the dissociation constant for ATP at the low affinity sites was approached through the ability of ATP to modify the rate of photo-oxidation of the enzyme in the presence of methylene blue; a value of 0.4 mM was obtained. The temperature dependence of the Km values in these two concentration ranges also differed markedly, and the estimated entropy of binding was +27 cal/degree per mol at the high affinity sites, whereas it was -20 cal/degree per mol at the low affinity sites. Moreover, the relative affinities of various congeners of ATP as of the K+ -dependent phosphatase reaction of the enzyme indicated an interaction at the low-affinity sites for ATP: ATP, ADP, CTP, and the [beta-gamma] -imido analog of ATP all competed with Ki values near those for the ATPase reaction at the low affinity sites. Conversely, the Km for nitrophenyl phosphate as a substrate for the phosphatase reaction was near its Ki as a competitor at the low-affinity sites of the ATPase reaction. These observations are incorporated into a reaction scheme with two classes of substrate sites on a dimeric enzyme, manifesting idverse enzymatic and transport characteristics.     

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.     

Inhibition of erythrocyte membrane (Ca2+ + Mg2+)-ATPase by the organophosphorus insecticides parathion and methylparathion

Organophosphorus insecticides parathion and methylparathion non-competitively inhibited the activity of (Ca²⁺ + Mg²⁺)-ATPase bound to and solubilized from pig erythrocyte membrane. Both enzyme preparations exhibited biphasic substrate curves displaying the existence of two functional active sites with low and high affinity to ATP. Also, the relationship between the activity of bound enzyme and Ca²⁺ concentration was biphasic. The activity reached maximum at 20 μM then dropped progressively as the Ca²⁺ concentration was raised. The inhibition of the activity was more pronounced for parathion than for methylparathion and the solubilized enzyme preparation was more affected than the bound one. The inhibition constants (K_i) for parathion for bound enzyme were 55 and 158 μM for high- and low-affinity active sites, respectively; for methylparathion these values equalled 74 and 263 μM, respectively. K_i values for parathion were 36 and 118 μM for solubilized enzyme (high- and low-affinity sites, respectively), for methylparathion −62 and 166 μM, respectively. The magnitude of the effect was greater for a low Ca²⁺ concentration, which could arise from different conformational states of the enzyme at different calcium concentrations. The results of the experiment suggest that the insecticides inhibited the ATPase by binding to a site on the enzyme rather than by the interaction with associated lipids, although lipids could weaken the action of the compounds due to the strong affinity of organophosphorus insecticides to lipids.     

Mechanisms of detergent effects on membrane-bound (Na+ + K+)-ATPase

Because the nonionic detergent octaethylene glycol dodecyl ether has been used extensively for studies on active solubilized preparations of (Na+ + K+)-ATPase, we tried to see if the detergent alters the properties of the membrane-bound enzyme prior to solubilization. Addition of the detergent, at concentrations below its critical micellar concentration, to reaction mixtures containing the highly purified membrane-bound enzyme reduced the K0.5 of ATP for (Na+ + K+)-dependent ATPase activity without affecting the maximal velocity or abolishing the negative cooperativity of the substrate-velocity curve. Under these conditions, however, the enzyme was not solubilized as evidenced by complete sedimentation of the membrane fragments containing the enzyme upon centrifugation at 100,000 X g for 30 min. Other nonsolubilizing effects of the detergent included an increase in K0.5 of K+, inhibition of Na+-dependent ATPase with no effect on K0.5 of ATP for this activity, and reductions in the spontaneous decomposition rates of the K+-sensitive phosphoenzyme obtained from ATP and the phosphoenzyme obtained from Pi. The nonsolubilizing effects of the detergent on the purified enzyme were obtained with no detectable lag, were readily reversible, and could be distinguished from its vesicle-opening effects on crude membrane preparations. Several other nonionic and ionic detergents had similar effects on the enzyme. The findings indicate (a) detergent binding to hydrophobic sites on extramembranous segments of enzyme subunits; (b) that occupation of these sites mimics the effects of ATP at a low-affinity regulatory site with no effect on high-affinity ATP binding to the catalytic site; and (c) that in studies on detergent-solubilized preparations, it is necessary to distinguish between the effects of solubilization per se and detergent effects at the regulatory site.     

(Na+,K+)-ATPase: a new assay of Na+-ATPase reveals covert anti-pump antibodies

A new assay is described for rat (Na⁺,K⁺)-ATPase [EC 3.6.1.3] prepared from renal medullary or crude liver membranes. With ATP at 1 μm, initial rates of ouabain-sensitive decreases in substrate concentrations are followed by measuring diminished ATP-driven luciferin-luciferase light production. Under these conditions, using highly purified enzyme preparations, Na⁺ and K⁺ ions stimulate and inhibit initial ATP hydrolysis rates, respectively. Therefore, it is likely that the assay measures Na⁺-ATPase partial reactions of the pump. A monospecific polyclonal rabbit anti-rat pump antiserum blocks Na⁺-dependent ATPase measured with the luciferase-linked ATPase assay, whereas conventional assays of purified pump activity at 3.0 mm ATP fail to reveal immunochemical blockade.     

A microsomal ATP-activated pyridine nucleotide transhydrogenase

An ATP-activated transhydrogenase which catalyzes the reduction of TPN⁺ by DPNH has been demonstrated in the microsomal fraction from the endosperm of immature Echinocystis macrocarpa seeds. The activity is specifically dependent on the presence of ATP (K_m of approximately 0.1 mm) of several nucleotides tested. The reaction is stimulated by MgCl₂ addition up to concentrations of 6 mm. When 10^?2m EDTA is added to the assay mixture in the absence of added MgCl₂, a transhydrogenation reaction is observed which no longer shows any dependence on added ATP. A TPN⁺-dependent ATPase activity can be demonstrated in these preparations, but no fixed stoichiometry between ATP cleavage and TPNH formation could be established. A lag in attaining the maximal rate of transhydrogenation is seen unless the enzyme is preincubated for 10 min with ATP before initiating the reaction. It can further be shown that preincubation of the enzyme with ATP followed by removal of the ATP on a Dowex 1 column produces an enzyme capable of catalyzing the transhydrogenation without the further addition of ATP. 2,4-Dinitrophenol and thyroxin are effective inhibitors of the transhydrogenase and 2,4-dinitrophenol was shown to inhibit the activating effect of ATP during the preincubation period. It is concluded that the role of ATP is in the modification of the enzyme rather than direct participation in the transhydrogenation. The transhydrogenase is inhibited by ADP and AMP. This results in a response of the enzyme to adenylate energy charge in a manner characteristic for regulatory enzymes which participate in ATP-utilizing metabolic sequences.     

Modification of (Na+ + K+)-dependent ATPase by fluorescein isothiocyanate: evidence for the involvement of different amino groups at different PH values

Fluorescein isothiocyanate (FITC) reactivity with the (Na⁺ + K⁺)-ATPase was studied at pH 6.5 and 9.0. Reaction with FITC is nearly complete in 30 min and is irreversible at both pH values. Differential inhibition of enzyme activity is observed at the two pH values as follows: at pH 6.5 the maximal inhibition reached is only 35–45% of the ATPase or p-nitrophenylphosphatase activities, whereas at pH 9.0 ATPase activity can be completely inhibited while maximal phosphatase inhibition is ca. 50%. At all concentrations of FITC tested, more FITC is incorporated into the enzyme at pH 9.0 than at 6.5. At both pH values NaCl increases the inhibition due to FITC while KCl protects against the inhibition. ATP protects the enzyme at both pH values with a K_0.5 in the range of 8–20 μm. Enzyme that is partially inactivated at either pH shows no significant change in the K_0.5 values for Na⁺ or K⁺ or in the K_{m app} for ATP or p-nitrophenylphosphate for the remaining activity. The binding of ⁴⁸VO₄ is not changed by reaction with FITC at either pH, while [³H]ouabain binding is inhibited after reaction at pH 9.0 only in the presence of Mg⁺² + Na⁺ + ATP. [³H]Ouabain binding in the presence of Mg⁺² + inorganic phosphate is not inhibited by FITC reaction. Enzyme reacted at both pH values exhibits the expected fluorescein fluorescence (λ_ex = 490, λ_em = 520) but only with enzyme reacted at pH 9.0 is fluorescence quenching by K⁺ or reversal by Na⁺ observed. These results suggest that different classes of amino groups react with FITC at the two pH values tested, and that these groups have distinct roles in the different activities of the enzyme.     

Studies on the heat-precipitated phosphoenzyme complex of eel electric organ (Na + K).ATPase.

When the hydrolytic reaction between eel electric organ (Na + K) · ATPase and [γ-³²P]ATP is terminated at neutral pH by heat precipitation, a phosphoenzyme complex is formed which reaches maximal levels in the simultaneous presence of Mg, Na, and K. After formation of a steady-state level of phosphoenzyme in the presence of Mg and Na, a pulse of K increases the level of the heat-precipitated phosphoenzyme (while decreasing the level of the acid-precipitated phosphoenzyme). The formation of the heat-precipitated phosphoenzyme is clearly inhibited by ouabain only when the phosphoenzyme is formed in the presence of Mg, Na, and K. Inorganic phosphate decreases the level of the heat-precipitated phosphoenzyme, but not that of the acid-precipitated phosphoenzyme (in the presence of Mg and Na or in the presence of Mg, Na, and K). Moreover, a heat-precipitated, ouabain-sensitive phosphoenzyme forms in the reaction between the eel (Na + K) · ATPase and ³²P_i with or without ATP. The pH stability of the heat-precipitated phosphoenzyme complex is maximal at pH 6 to 8, and this complex shows little or no reactivity with neutral hydroxylamine, suggesting that the phosphate is not bound to an acyl residue of the protein. These experiments indicate that both heat-resistant and acid-resistant phosphoenzymes are formed during the (Na + K) · ATPase reaction at pH 7.4.     

Characterization of conformational changes in (Na,K) ATPase labeled with fluorescein at the active site

Conformational changes have been studied in (Na,K) ATPase labeled at or near the ATP binding region with fluorescein following incubation with fluorescein isothiocyanate (FITC). One or two fluorescein groups are bound per ATPase molecule. (Na,K) ATPase activity, phosphorylation from ATP, and nucleotide binding are abolished in labeled enzyme, but phosphorylation from inorganic phosphate or K-phosphatase activity are only partially inactivated. The fluorescein groups are incorporated only into the 96 KD catalytic chain of the (Na,K) ATPase, and presence of ATP during the incubation with FITC protects against the incorporation and inhibition of enzymic activity. Upon trypsin treatment of labeled membranes the fluorescein appears first in a 58 KD fragment and eventually is released into the medium. The fluorescein-labeled (Na,K) ATPase shows a large quenching of fluorescence (15–20%) on conversion of the E₁ or E₁ · Na conformation in cation-free or Na⁺-rich media to the E₂ · (K) form in K⁺ (or congeners Tl⁺, Rb⁺, Cs⁺, NH ₄ ⁺ ) rich media. Cation titrations suggest that K⁺ and Na⁺ ions compete at a single binding site and stabilize E₁ · Na or E₂ · (K) respectively;K _K0.23 mM,K _Na1.2 mM. The rate of the conformational transition E₂ · (K) E₁ · Na is slow,k=0.3 sec^–1, but contrary to previous experience [7, 8] ATP does not stimulate this rate. The rate of the transitions E₁ + K⁺ E₂ · (K) rises sharply with K⁺ concentration and shows saturation behavior, from which ak _max286 sec^–1 andK _k74 mM are deduced. The data support and extend the previous suggestion that K⁺ ions bound initially at a low-affinity (probably cytoplasm oriented) site in state E₁ are trapped in the occluded form E₂ · (K) by the conformational change poised far (K _c1000) in the direction of E₂ · (K). It is proposed in addition that at least two binding sites for K⁺ exist at the cytoplasmic surface of isolated (Na,K) ATPase in state E₁ but a large difference in affinities precludes detection in fluorescence titrations of more than one site. A variety of ligands in addition to K⁺ produce fluorescence-quenched or E₂ forms of the labeled (Na,K) ATPase. These include Mg²⁺ plus inorganic phosphate, without or with K⁺ ions (E₂P or E₂P · K) or with ouabain (E₂-ouabain or E₂P · ouabain). Na⁺ ions antagonize these effects. The collected data support the notion that there may be many subspecies of the E₁ and E₂ forms (either phosphorylated or nonphosphorylated) with different numbers of Na⁺ and/or K⁺ ions bound or occluded, each subspecies having a characteristic ability to catalyze reactions and/or transport cations. The relationship between the conformational changes in fluorescein-labeled enzyme and the subunit structure of the (Na,K) ATPase is discussed with particular reference to half of the site models for ATP hydrolysis.     

Harmaline: A competitive inhibitor of Na Ion in the (Na++K+)-ATPase system

Summary Experimental evidence is given that the hallucinogen harmaline (HME) behaves as an inhibitor of the (Na⁺+K⁺)-ATPase system, specifically in the Na⁺-dependent phosphorylation reaction. HME at 0.3 to 3mm inhibited several membrane ATPase preparations such as those from human erythrocytes, rat brain and squid retinal axons. The same concentration blocked Na⁺ outflow from squid giant axons. The behavior of several harmane derivatives such as harmine, harmalol and harmaline demonstrated that certain groups influenced the concentration for 50% inhibition of the ATPase system. The following evidence demonstrated that HME blocked the formation of the phosphorylated intermediate by competition with Na ions in the (Na⁺+K⁺)-ATPase reaction in rat brain. (1) The HME effect on the overall (Na⁺+K⁺)-ATPase reaction showed a fully competitive inhibition with respect to Na ion concentration. (2) The inhibition of the Na⁺-stimulated phosphorylation by HME was fully competitive with respect to Na ions, with or without oligomycin present. (3) HME inhibited the effect of ADP on the phosphorylation reaction using³²P-ATP. (4) HME did not accelerate the rate of membrane dephosphorylation by means of³²P-ATP and cold ATP.From the behavior of HME as a competitive inhibitor at Na ion sites of the (Na⁺+K⁺)-ATPase reactions one may gain information about (a) The chemical nature of Na⁺ sites which may be responsible for the selectivity of this cation, and (b) The sequence of Na⁺ and ATP entrance into the Na⁺-dependent phosphorylation reaction. The experimental evidence supports the hypothesis that the entrance of Na⁺ into the enzyme system may precede the formation of the phosphorylated intermediate.     

Tryptic digestion of the (Na + K)-ATPase is both sensitive to and modifies K+ interactions with the enzyme

Tryptic digestion of the (Na + K)-ATPase in the presence of choline chloride or NaCl (Na-type) and in the presence of KCl (K-type) produced distinct patterns of peptide fragments and losses of catalytic activity. TheK _0.5 for K⁺ to shift digestion from the Na-type, and its sensitivity to dimethyl sulfoxide and Triton X-100, were consistent with K⁺ acting at sites on the cytoplasmic face of the enzyme through which the K-phosphatase reaction also is activated. Reagents favoring the E₁ conformational states, oligomycin, Triton, and ATP, shifted the pattern toward the Na-type, whereas those favoring E₂ states, dimethyl sulfoxide, MgCl₂, and MnCl₂, shifted the pattern toward the K-type. Na-type digestion caused a greater loss of K-phosphatase than (Na + K)-ATPase activity, and the residual K-phosphatase activity was more sensitive to inhibition by Triton and ATP but stimulated more by dimethyl sulfoxide and inhibited less by P_i and MnCl₂; all these effects are consistent with such digestion shifting equilibria toward E₁ enzyme states. Accordingly, theK _0.5 for K⁺ to activate the (Na + K)-ATPase was increased. However, theK _0.5 for the K-phosphatase was unchanged; this observation requires revision of previous formulations, and bears on additional aspects of enzyme activity as well.     

Ca2+-Dependent activities of (Na+ + K+)-ATPase

Experiments on the effects of varying concentrations of Ca²⁺ on the Mg²⁺ + Na⁺-dependent ATPase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase showed that Ca²⁺ was a partial inhibitor of this activity. When Ca²⁺ was added to the reaction mixture instead of Mg²⁺, there was a ouabain-sensitive Ca²⁺ + Na⁺-dependent ATPase activity the maximal velocity of which was 30 to 50% of that of Mg²⁺ + Na⁺-dependent activity. The apparent affinities of the enzyme for Ca²⁺ and CaATP seemed to be higher than those for Mg²⁺ and MgATP. Addition of K⁺, along with Ca²⁺ and Na⁺, increased the maximal velocity and the concentration of ATP required to obtain half-maximal velocity. The maximal velocity of the ouabain-sensitive Ca²⁺ + Na⁺ + K⁺-dependent ATPase was about two orders of magnitude smaller than that of Mg²⁺ + Na⁺ + K⁺-dependent activity. In agreement with previous observations, it was shown that in the presence of Ca²⁺, Na⁺, and ATP, an acid-stable phosphoenzyme was formed that was sensitive to either ADP or K⁺. The enzyme also exhibited a Ca²⁺ + Na⁺-dependent ADP-ATP exchange activity. Neither the inhibitory effects of Ca²⁺ on Mg²⁺-dependent activities, nor the Ca²⁺-dependent activities were influenced by the addition of calmodulin. Because of the presence of small quantities of endogenous Mg²⁺ in all reaction mixtures, it could not be determined whether the apparent Ca²⁺-dependent activities involved enzyme-substrate complexes containing Ca²⁺ as the divalent cation or both Ca²⁺ and Mg²⁺.