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• W1964743049 abstract "The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Cardiotonic steroids like ouabain used in the treatment of congestive heart failure and arrhythmias specifically inhibit the Na,K-ATPase, and the detailed structure of the highly conserved binding site has recently been described by the crystal structure of the shark Na,K-ATPase in a state analogous to E2·2K+·Pi with ouabain bound with apparently low affinity (1Ogawa H. Shinoda T. Cornelius F. Toyoshima C. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 13742-13747Crossref PubMed Scopus (273) Google Scholar). In the present work inhibition, and subsequent reactivation by high Na+, after treatment of shark Na,K-ATPase with various metal fluorides are characterized. Half-maximal inhibition of Na,K-ATPase activity by metal fluorides is in the micromolar range. The binding of cardiotonic steroids to the metal fluoride-stabilized enzyme forms was investigated using the fluorescent ouabain derivative 9-anthroyl ouabain and compared with binding to phosphorylated enzyme. The fastest binding was to the Be-fluoride stabilized enzyme suggesting a preformed ouabain binding cavity, in accord with results for Ca-ATPase where Be-fluoride stabilizes the E2-P ground state with an open luminal ion access pathway, which in Na,K-ATPase could be a passage for ouabain. The Be-fluoride stabilized enzyme conformation closely resembles the E2-P ground state according to proteinase K cleavage. Ouabain, but not its aglycone ouabagenin, prevented reactivation of this metal fluoride form by high Na+ demonstrating the pivotal role of the sugar moiety in closing the extracellular cation pathway. The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Cardiotonic steroids like ouabain used in the treatment of congestive heart failure and arrhythmias specifically inhibit the Na,K-ATPase, and the detailed structure of the highly conserved binding site has recently been described by the crystal structure of the shark Na,K-ATPase in a state analogous to E2·2K+·Pi with ouabain bound with apparently low affinity (1Ogawa H. Shinoda T. Cornelius F. Toyoshima C. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 13742-13747Crossref PubMed Scopus (273) Google Scholar). In the present work inhibition, and subsequent reactivation by high Na+, after treatment of shark Na,K-ATPase with various metal fluorides are characterized. Half-maximal inhibition of Na,K-ATPase activity by metal fluorides is in the micromolar range. The binding of cardiotonic steroids to the metal fluoride-stabilized enzyme forms was investigated using the fluorescent ouabain derivative 9-anthroyl ouabain and compared with binding to phosphorylated enzyme. The fastest binding was to the Be-fluoride stabilized enzyme suggesting a preformed ouabain binding cavity, in accord with results for Ca-ATPase where Be-fluoride stabilizes the E2-P ground state with an open luminal ion access pathway, which in Na,K-ATPase could be a passage for ouabain. The Be-fluoride stabilized enzyme conformation closely resembles the E2-P ground state according to proteinase K cleavage. Ouabain, but not its aglycone ouabagenin, prevented reactivation of this metal fluoride form by high Na+ demonstrating the pivotal role of the sugar moiety in closing the extracellular cation pathway. The Na,K-ATPase is indispensable in maintaining cellular homeostasis in animals. This integral membrane protein is an ion pump fueled by ATP and responsible for maintaining electrochemical gradients for Na+ and K+ across animal cell membranes, which is essential for many physiological processes like secondary active co- and counter-transport, volume regulation, and forms the basis for generating the resting membrane potential. In sarco(endo)plasmic Ca2+-ATPase from skeletal muscle (SERCA1a), 2The abbreviations used are: SERCA1asarco(endo)plasmic Ca-ATPase 1aOouabainAO9-anthroyl ouabainOGouabageninMeFmetal fluoride complexCTScardiotonic steroidPKproteinase KEPphosphoenzyme. another representative member of P-type ATPases, metal fluorides are potent inhibitors of ATPase activity, binding to the enzyme phosphorylation site (D351) as analogues of phosphate (2Missiaen L. Wuytack F. De Smedt H. Vrolix M. Casteels R. Biochem. J. 1988; 253: 827-833Crossref PubMed Scopus (56) Google Scholar, 3Murphy A.J. Coll R.J. J. Biol. Chem. 1992; 267: 16990-16994Abstract Full Text PDF PubMed Google Scholar, 4Murphy A.J. Coll R.J. J. Biol. Chem. 1993; 268: 23307-23310Abstract Full Text PDF PubMed Google Scholar, 5Troullier A. Giradet J.L. Dupont Y. J. Biol. Chem. 1992; 267: 22821-22829Abstract Full Text PDF PubMed Google Scholar). sarco(endo)plasmic Ca-ATPase 1a ouabain 9-anthroyl ouabain ouabagenin metal fluoride complex cardiotonic steroid proteinase K phosphoenzyme. In Ca-ATPase various structural analogues of enzyme phosphoforms (EP) have been stabilized and structurally characterized using fluoride analogues of phosphate. Thus, complexes of magnesium, aluminum, and beryllium with fluoride (MgFx, AlFx, and BeFx) stabilize analogues of the E2·P product state (E2·MgF42−), the E2∼P transition state (E2·AlF4−), and the E2-P ground state (E2·BeF3−), respectively (reviewed in Ref. 6Toyoshima C. Arch. Biochim. Biophys. 2008; 476: 3-11Crossref PubMed Scopus (182) Google Scholar). Inclusion of ADP together with AlFx stabilizes an E1∼P·ADP form (7Toyoshima C. Nomura H. Tsuda T. Nature. 2004; 432: 361-368Crossref PubMed Scopus (382) Google Scholar, 8S⊘rensen T.L. M⊘ller J.V. Nissen P. Science. 2004; 304: 1672-1675Crossref PubMed Scopus (373) Google Scholar). In Na,K-ATPase similar effects of various fluoride analogues are found (9Robinson J.D. Davis R.L. Steinberg M. J. Bioenerg. Biomemb. 1986; 18: 521-531Crossref PubMed Scopus (39) Google Scholar, 10Murphy A.J. Hoover J.C. J. Biol. Chem. 1992; 267: 16995-17000Abstract Full Text PDF PubMed Google Scholar), but are less well characterized. Recently, Na,K-ATPase from pig kidney and shark rectal glands with 2 K+-ions, or Rb+-ions, in the cation binding site, and MgF42− in the phosphorylation site has been crystallized (11Morth J.P. Pedersen B.P. Toustrup-Jensen M.S. S⊘rensen T.L. Petersen J. Andersen J.P. Vilsen B. Nissen P. Nature. 2007; 450: 1043-1049Crossref PubMed Scopus (705) Google Scholar, 12Shinoda T. Ogawa H. Cornelius F. Toyoshima C. Nature. 2009; 459: 446-450Crossref PubMed Scopus (492) Google Scholar), which is presumed to represent the E2·2K+·Pi state. Cardiotonic steroids (CTSs) like ouabain are specific inhibitors of the Na,K-ATPase (13Schatzmann H.J. Helv. Physiol. Pharmacol. Acta. 1953; 11: 346-354PubMed Google Scholar). This is the basis for the therapeutic action of CTSs like digoxin and digitoxin in treatment of congestive heart failure and arrhythmia (14Lingrel J.B. Annu. Rev. Physiol. 2010; 72: 395-412Crossref PubMed Scopus (234) Google Scholar). Inhibition of myocardial Na,K-ATPase leads to elevated intracellular Na+-concentration, which suppresses NCX, the 3Na+-Ca2+ exchanger, thus increasing intracellular Ca2+ and producing an increase in the contractility of the heart and cardiac output (the inotropic effect). Ouabain binds to the extracellular side of the Na,K-ATPase and mainly to a phosphorylated intermediate (15Yoda A. Yoda S. Mol. Pharmacol. 1982; 22: 700-705PubMed Google Scholar); however, it is still not known to which intermediate ouabain principally binds during enzyme turnover. The ouabain binding site was recently determined for the shark enzyme in an apparent low affinity E2·2K+·Pi form (1Ogawa H. Shinoda T. Cornelius F. Toyoshima C. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 13742-13747Crossref PubMed Scopus (273) Google Scholar). A low-resolution (∼5 Å) crystal structure of kidney Na,K-ATPase in the E2P state indicates that ouabain is bound in a similar position, but likely with high affinity (16Yatime L. Laursen M. Morth J.P. Esmann M. Nissen P. Fedosova N.U. J. Struct. Biol. 2011; 174: 296-306Crossref PubMed Scopus (136) Google Scholar). In the present study interactions of various metal fluorides (MeF), acting as phosphate analogues, with shark Na,K-ATPase are investigated and the binding of ouabain and its aglycone ouabagenin to various fluoride analogues of EP-intermediates of Na,K-ATPase is characterized to elucidate structural constraints for binding of CTS to various EP phosphoforms, especially regarding the importance of the sugar moiety of CTS. The cardiotonic steroids ouabain and ouabagenin (OG), MgCl2, BeSO4, AlCl3, and NaF were from Sigma. 9-Anthroyl ouabain (AO) was from Invitrogen (Carlsbad, CA). Crude membrane fractions (microsomes) from the rectal gland of the shark Squalus acanthias were prepared by homogenization followed by washing and isolation by centrifugation in 30 mm histidine, 1 mm EDTA, 0.25 m sucrose, pH 6.8. The purified microsomes were activated by a mild DOC treatment (∼0.15% DOC) to extract extrinsic proteins and to open sealed vesicles. After washing and resuspension the purified membrane preparation was obtained by differential centrifugation essential as previously described (17Skou J.C. Esmann M. Methods in Enzymology. 1988; 156: 43-46Crossref PubMed Scopus (27) Google Scholar). The preparation was suspended in histidine/EDTA buffer with 25% glycerol and kept at −20 °C. The preparation had a specific hydrolytic activity of ∼30 units/mg at 37 °C and contained the α1-, β1-subunits together with the FXYD10 regulatory subunit (18Mahmmoud Y.A. Vorum H. Cornelius F. J. Biol. Chem. 2000; 275: 35969-35977Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Protein concentrations, ranging from 3–5 mg/ml, were determined using Peterson's modification (19Peterson G.L. Methods Enzymol. 1983; 91: 95-119Crossref PubMed Scopus (1142) Google Scholar) of the Lowry method (20Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), using bovine serum albumin as a standard. The specific enzyme activity was measured using either the Fiske and SubbaRow method (21Fiske C.H. SubbaRow Y. J. Biol. Chem. 1925; 66: 375-400Abstract Full Text PDF Google Scholar) with Amidol as the reducing agent, or the more sensitive method of Baginsky et al. (22Baginski E.S. Foa P.P. Zak B. Clin. Chim. Acta. 1967; 14: 155-158Crossref Scopus (332) Google Scholar). The activity was measured at 23 °C in a test medium containing 130 mm NaCl, 20 mm KCl, 4 mm MgCl2, 3 mm Tris-ATP, and 0.33 mg/ml bovine serum albumin. Histidine or imidazole (30 mm) was used as buffer, depending on pH. The inhibition of Na,K-ATPase activity by MgFx by equilibrium binding was characterized by mixing 5 mm MgCl2 and increasing concentrations of NaF (1–100 mm) in imidazole 30 mm pH 6.5, 7.5, or 8.5 followed by addition of Na,K-ATPase and preincubation for 10 min. at 23 °C. The inhibition by BeFx and AlFx was performed by mixing 5 mm NaF and increasing concentrations of BeSO4 or AlCl3 (1–200 μm) followed by preincubation with the enzyme, as described above. The reaction with AlFx·ADP was produced by including 1 mm ADP in the AlFx reaction media. Following preincubation, the Na,K- ATPase activity at 23 °C was determined at optimal turnover conditions, i.e. in 130 mm Na+, 20 mm K+, 4 mm Mg2+, 3 mm ATP, and 30 mm imidazole (pH 7.5). The time course of inhibition at various concentrations of fluorides was determined by varying the time of preincubation with fluorides (15–180 s) as described above, followed by addition of the enzyme activity test medium. After 1 min, the reaction was stopped by 50% TCA and liberated Pi was determined. The rate of inhibition induced by binding of CTS to non-phosphorylated or MgPi-phosphorylated Na,K-ATPase was determined by preincubation of enzyme in 30 mm imidazole (pH 7.5) with either 5 mm Mg2+, or 5 mm Mg2+ + 1 mm Pi with 1 μm ouabain, ouabagenin, or anthroyl ouabain for varying time periods followed by measurement of hydrolytic activity. The enzyme was first reacted with metal fluoride complexes by incubating in 5 mm NaF (or KF), 30 mm imidazole pH 7.5 and either 5 mm MgCl2, 5 μm BeSO4, 200 μm AlCl3, or 200 μm AlCl3 plus 1 mm ADP for 10 min. at 23 °C to obtain maximum inhibition. Then 150 mm NaCl was added to the enzyme and the hydrolytic activity measured at different time intervals (0–60 min). The reactivation of metal fluoride-treated enzyme reacted with ouabain or ouabagenin was tested by including the CTS in the metal fluoride reaction medium for 50 min after the initial 10 min preincubation with metal fluoride followed by addition of 150 mm NaCl and measurement of hydrolytic activity. Fluorescence associated with 9-anthroyl ouabain (AO) binding to the Na,K-ATPase was measured using a Spex Fluorolog-3 spectrofluorometer (Horiba Jobin Yvon). Excitation wavelength was set at 370 nm using a band-pass of 5 nm, and the emission wavelength was 480 nm with band-pass 10 nm. The sample was placed in a thermostated cuvette (20 °C) with magnetic stirring and contained 30 mm Tris-buffer pH 7.0, 4 mm MgCl2, and 1 μm anthroyl ouabain. Before fluorescence measurements the enzyme was incubated with 4 mm MgCl2, 4 mm Pi to produce the E2P-state, or with different metal fluorides as indicated above to produce the enzyme complexes E·MgFx, E·BeFx, E·AlFx, and E·AlFx·ADP. Enzyme was incubated for 3 min at 20 °C followed by incubation on ice for 1 h before measurements. To measure AO fluorescence ∼60 μg of the incubated enzyme was added to the cuvette in a final volume of 2 ml with 1 μm AO. Baseline fluorescence was measured with enzyme preincubated with 1 mm ouabain. Controlled proteolysis of the shark rectal gland α-subunit was performed in a reaction mixture containing 100 μg of protein suspended in 25 mm histidine pH 7.0, and the following ligands to stabilize the enzyme in specific conformations: 4 mm NaF and 4 mm MgCl2 (E·MgFx), 4 mm NaF, and 50 μl BeSO4 (E·BeFx), 4 mm NaF, and 100 μm AlCl3 (E·AlFx), or 4 mm NaF and 100 μm AlCl3 plus 1 mm ADP (E·AlFx·ADP). The reaction was initiated by the addition of 2 μg of proteinase K for 40 min at 20 °C and terminated with SDS sample buffer containing 1% trichloroacetic acid to irreversibly inhibit the protease. 40 μg of protein was loaded onto 8% SDS-PAGE, and the gel was stained with Coomassie Blue. Identification of cleavage products was performed by Edman degradation analysis (Alphalyse, Odense, DK). Results are expressed as mean ± S.E. Inhibition by fluorides was evaluated by fitting to a sigmoid dose-response equation (Hill Equation 1) using the GraphPad program Prism 5,y=y0+ymax−y01+10Log(Ki−X)nH(Eq. 1) where y0 and ymax are baseline and maximum activity and x is the concentration of inhibitor. Ki is the inhibitor concentration that gives 50% inhibition and nH is the Hill coefficient. The time course of fluoride inhibition or AO fluorescence was fitted with one phase exponential Equation 2,y=(y∞−y0)e−kobst+y0(Eq. 2) where kobs is the observed rate constant and y0 and y∞ are the initial and final values. The relative intensity of inhibition or fluorescence is then Equation 3.y∞−y0y0(Eq. 3) Comparison between best-fit values was performed using an F test and p < 0.05 was considered significant. Structural figures were prepared using PyMol. Inhibition of Na,K-ATPase activity by increasing concentration of NaF at a constant MgCl2 concentration of 5 mm is demonstrated in Fig. 1 at pH 6.5, 7.5, and 8.5. The data were fitted with a Hill equation and showed an increase in the NaF inhibitor constant (Ki) as the pH is increased. Thus Ki increased from 1.1 mm to 6.4 mm when pH increased from 6.5 to 8.5. The Hill coefficient (shared at the different pH values) was −2.2 and significantly different from −1. As the Hill coefficient is empirical and provides only lower limits on the binding stoichiometry, this is in accordance with 4 fluorine atoms present in the stable complex MgF42− in the recent crystal structure of shark Na,K-ATPase (12Shinoda T. Ogawa H. Cornelius F. Toyoshima C. Nature. 2009; 459: 446-450Crossref PubMed Scopus (492) Google Scholar). There was no systematic variation in the Hill coefficient with pH indicating that the coordination number for fluoride did not change within this pH range, as indicated in other nucleotide-binding proteins (23Schlicting I. Reinstein J. Nature. 1999; 6: 721-723Google Scholar). Equilibrium binding of MgFx, BeFx, AlFx, and AlFx·ADP to Na,K-ATPase was evaluated by measuring the inhibition of specific ATPase activity after preincubation for 10 min at 23 °C in solutions containing 5 mm NaF and increasing concentrations of MgCl2, BeSO4, AlCl3, or AlCl3 + 1 mm ADP. Fig. 2 shows inhibition by MgCl2 (panel A), BeSO4 (panel B), AlCl3 (panel C), or AlCl3 + ADP (panel D), all at 5 mm NaF and pH 7.5. The data were fitted with a Hill equation and the inhibitor dissociation constant and Hill coefficient evaluated. The Hill coefficient was significantly different from −1 at all conditions indicating inhibition by binding of more than one metal ion in the phosphorylation domain. In the case of MgCl2 this is in accordance with the binding of two Mg2+ ions in the phosphorylation domain, one with MgF42− near D376 and one at the Mg2+ subsite near D717 in shark, Fig. 3 (12Shinoda T. Ogawa H. Cornelius F. Toyoshima C. Nature. 2009; 459: 446-450Crossref PubMed Scopus (492) Google Scholar). From the present data it seems likely that also beryllium and aluminum will bind to the Mg2+ subsite in the absence of Mg2+. Beryllium and aluminum have not previously been identified in the P-domain (D703) of SERCA crystal structures (see Ref. 8S⊘rensen T.L. M⊘ller J.V. Nissen P. Science. 2004; 304: 1672-1675Crossref PubMed Scopus (373) Google Scholar), and identification by crystallography will probably be difficult due to lack of strong anomalous signals from these metals. The inhibitor constants measured at pH 6.5, 7.5, and 8.5 are given in Table 1. As indicated the inhibitor affinity increased with decreasing pH and the inhibitor affinity was highest for BeFx followed by AlFx, AlFx·ADP, and MgFx. This order did not change with pH. The inhibition by AlFx·ADP was much less pH-sensitive, as seen from Table 1. No systematic variation in the fitted Hill coefficients was present at the different pH values, and the Hill coefficient was therefore shared in the fitting procedure to data at the various pH values.FIGURE 3Coordination of MgF42−, a Pi analog, and of a Mg2+ ion at the phosphorylation site in the crystal structure of Na,K-ATPase from shark rectal gland in a state representing E2·2K+·Pi. In this state the Pi analog MgF42− is coordinated by residues in the phosphorylation (P) domain, including the phosphorylated residue D376. A divalent metal ion, in this case Mg2+ is located between D376 and another critical residue D717. Orange dotted lines represent coordination of ligands, and green dotted lines show hydrogen bonds likely to be important in stabilizing coordinating residues. Small red spheres represent water molecule. Coordinates of the atomic model are derived from PDB entry 2ZXE.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Inhibitor constant (Ki/μm) for various metal fluorides at different pH valuesKi/μmnH (shared)aThe value of the Hill coefficient in the fitting is shared between experiments performed at the three pH values. In all cases, the nH value was found to be significantly different from −1.0.pH 6.5pH 7.5pH 8.5MgCl239.7 ± 0.7133 ± 3708 ± 67−1.63 ± 0.07BeSO42.6 ± 0.22.5 ± 0.35.2 ± 0.2−1.41 ± 0.09AlCl38.5 ± 0.312.5 ± 0.226.4 ± 0.2−2.05 ± 0.15AlCl3·ADP39.5 ± 0.232.4 ± 0.342.6 ± 0.7−1.33 ± 0.11a The value of the Hill coefficient in the fitting is shared between experiments performed at the three pH values. In all cases, the nH value was found to be significantly different from −1.0. Open table in a new tab In Fig. 4A the time course of Na,K-ATPase inhibition induced by adding enzyme to a premix of 5 mm MgCl2 and increasing concentrations of NaF (1–100 mm) at pH 7.5 is illustrated. The data could be satisfactorily fitted with a mono-exponential relation with an observed rate constants, kobs. In Fig. 4B the kobs is plotted against the concentration of NaF. The relation is steeply sigmoid and the data are compatible with a Hill equation where the K0.5 is 42 mm when nH is set to 4, in accordance with 4 fluorine atoms in the metal fluoride complex, MgF42−, that binds to the phosphorylation site (12 c.f. Fig. 3). At pH 7.5 the observed rate constant at saturating NaF (75 mm) was 0.13 s−1 comparable to an apparent second order rate constant of kapp = 1.7 s−1 m−1. These rather small values indicated that a slow isomerization step follows the initial collision interaction between the Mg2+-bound Na,K-ATPase (denoted E for simplicity) and metal fluoride (MeF), shown in Reaction Scheme I,in which KMeF is the MeF dissociation constant. In Fig. 5 the observed rate constant, kobs, for inhibition of Na,K-ATPase activity by increasing concentrations of BeSO4, AlCl3, and AlCl3 + ADP (1 mm) all in the presence of 5 mm NaF are shown. The concentrations of BeSO4 and AlCl3 were converted to concentrations of the metal fluoride complexes BeF3− and AlF4− as estimated to comprise ∼70 and 60% of the total metal fluoride complexes in the presence of 5 mm fluoride (24Goldstein G. Anal. Chem. 1964; 36: 243-244Crossref Scopus (59) Google Scholar). The relation between the rate of inhibition and concentration of MgCl2 in the presence of 5 mm NaF could not be measured accurately because of very low kobs of this reaction. As seen kobs was a hyperbolic function of the metal fluoride concentration in accordance with Reaction Scheme I, and not linear as expected for a one-step reaction. According to this reaction the following analytical expression, Equation 4, for the observed rate constant is given.kobs−k2[MeF]KMef+[MeF]+k−2(Eq. 4) Using this equation to fit the data shown in Figs. 5A-C KMeF, k2, and k−2 were found. Thus k−2 is the intercept with the y axis, and kobs at saturating metal fluoride concentration equals k−2+k2. The dissociation constants for BeF3−, AlF4−, and AlF4−·ADP at pH 7.5 found from the fits were: 358 μm, 149 μm, and 218 μm, respectively. As indicated all values of KMeF were significantly larger than the half-saturation inhibitor constants (Ki) calculated from equilibrium measurements of metal fluoride inhibition of enzyme activity shown in Fig. 2. This is expected, since KMeF will be larger than Ki by a factor of (k2 + k−2)/k−2 as indicated by the reaction given in scheme I. Taking this into account the calculated K0.5 (Ki) values at pH 7.5 became 9 μm for BeF3−, 17 μm for AlF4−, and 26 μm in the case of AlF4−·ADP, in reasonable agreement with the measured Ki value given in Fig. 2 given the uncertainty in the estimation of k−2.FIGURE 4Time course of NaF inhibition of Na,K-ATPase activity at increasing NaF-concentration and a constant MgCl2 concentration of 5 mm at pH 7.5. A, rate of inhibition at increasing NaF concentrations (1, 3, 10, 30, 50, 75, and 100 mm). The curves are one-phase exponential fits. B, observed rate constants, kobs as a function of the NaF concentration. The stippled curve shows the fitted Hill equation with a K0.5 values of 42 ± 4 mm and a Hill coefficient set to 4.0.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Fluoride concentration dependence of kobs for inhibition of Na,K-ATPase activity by different fluoride complexes at pH 7.5. A, BeF3−; B, AlF4−; C, AlF4−·ADP. The curves show fit of the equation kobs = (k2[MeF])/(KMeF + [MeF])) + k−2 to the data. The fitted k2, k−2, and KMeF values for BeF3− (panel A) are: 0.32 ± 0.14 s−1, 0.009 ± 0.004 s−1, and 358 ± 65 μm; for AlF4− (panel B): 0.16 ± 0.05 s−1, 0.008 ± 0.002 s−1, and 149 ± 25 μm; and for AlF4−·ADP (panel C): 0.070 ± 0.004 s−1, 0.009 ± 0.002 s−1, and 218 ± 63 μm. From these values, the calculated inhibitor constant Ki = KMeF/(k2+k−2)/k−2 then becomes 9 ± 4 μm for BeF3−, 17 ± 5 μm for AlF4−, and 26 ± 7 μm for AlF4−·ADP.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To characterize the various fluoride-stabilized phosphoform analogues proteinase K (PK) cleavage was employed. The proteinase K cleavage pattern of SERCA1a has previously been characterized in details to probe the protein folding (25Juul B. Turc H. Durand M.L. de Gracia A. Denoroy L. M⊘ller J.V. Champeil P. le Maire M. J. Biol. Chem. 1995; 270: 20123-20134Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 26Lenoir G. Picard M. Gauron C. Montigny C. Le Maréchal P. Falson P. le Maire M. M⊘ller J.V. Champeil P. J. Biol. Chem. 2004; 279: 9156-9166Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In Fig. 6A the PK cleavage patterns of enzyme stabilized by the various fluoride analogues of phosphate are shown and compared with the cleavage pattern of E2P produced by phosphorylation of the enzyme with MgPi. As seen the PK cleavage patterns for E·BeFx and E·MgPi (lanes 3 and 5) were very similar. In both cases a clear band with approximately molecular mass of 95 kDa was observed, which is missing or much weaker in all other conditions. This band is probably equivalent to the p95 band in SERCA obtained after PK cleavage in the absence of Ca2+ arising from cleavage at L119/K120 (25Juul B. Turc H. Durand M.L. de Gracia A. Denoroy L. M⊘ller J.V. Champeil P. le Maire M. J. Biol. Chem. 1995; 270: 20123-20134Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 26Lenoir G. Picard M. Gauron C. Montigny C. Le Maréchal P. Falson P. le Maire M. M⊘ller J.V. Champeil P. J. Biol. Chem. 2004; 279: 9156-9166Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In shark Na,K-ATPase this cleavage site is equivalent to F161/K162 positioned at a similar short helix (M2′) in the connection between M2 and the A-domain (as in SERCA), and previously demonstrated to be a trypsin cleavage site exposed by ouabain binding (27Cornelius F. Mahmmoud Y.A. Biochemistry. 2009; 48: 10056-10065Crossref PubMed Scopus (18) Google Scholar). The cleavage pattern of E·MgFx and E·AlFx appeared similar, both lacking the 95 kDa band, although the α subunit seemed to be somewhat protected from PK cleavage by AlFx. In Fig. 6 (panels B and C) the time course of PK cleavage of BeFx- and AlFx-treated enzyme is shown. As seen AlFx protected completely the cleavage site producing the 95 kDa band. In the presence of ADP (panel A, lanes 6 and 7) the PK cleavage pattern of enzyme treated with AlFx or MgPi changed, the band migrating at ∼55 kDa was much more prominent than in all other cleavage patterns, whereas the 30 kDa band was weaker. The latter band probably arises from secondary cleavage of the 55 kDa band (see panels B and C) and Edman degradation analysis indicated an N-terminal sequence 376DKTG that could be recognized in the shark Na,K-ATPase sequence as corresponding to the phosphorylation site indicating cleavage at S375 just next to the aspartate D376, the residue phosphorylated. Thus, this fragment seems to be equivalent to the p30 fragment in the PK cleavage of SERCA, which has a cleavage site at S350 immediately before the phosphorylation site and results from secondary cleavage of a p54 fragment containing M3/M4 together with a large part of the cytoplasmic phosphorylation domain (25Juul B. Turc H. Durand M.L. de Gracia A. Denoroy L. M⊘ller J.V. Champeil P. le Maire M. J. Biol. Chem. 1995; 270: 20123-20134Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In Fig. 7 the PK cleavage sites determined for the shark Na,K-ATPase and as identified in SERCA1a (25Juul B. Turc H. Durand M.L. de Gracia A. Denoroy L. M⊘ller J.V. Champeil P. le Maire M. J. Biol. Chem. 1995; 270: 20123-20134Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 26Lenoir G. Picard M. Gauron C. Montigny C. Le Maréchal P. Falson P. le Maire M. M⊘ller J.V. Champeil P. J. Biol. Chem. 2004; 279: 9156-9166Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) (translated to shark Na,K-ATPase numbering) are indicated in a schematic view (panel A) and in the ribbon model (panel B) of the shark Na,K-ATPase crystal structure (12Shinoda T. Ogawa H. Cornelius F. Toyoshima C. Nature. 2009; 459: 446-450Crossref PubMed Scopus (492) Google Scholar). According to this, the 55 kDa peptide includes amino acids E278 to I749/V762, and the 30 kDa peptide comprises D376 to V603. Although the sequence around the phosphorylation site is conserved between SERCA and Na,K-ATPase the PK digestion pattern was different in the two enzymes, because in SERCA this cleavage site is protected completely in all of the phosphoforms (28Danko S. Daiho T. Yamasaki K. Kamidochi M. Suzuki H. Toyoshima C. FEBS Lett. 2001; 489: 277-282Crossref PubMed Scopus (60) Google Scholar, 29Danko S. Yamasaki K. Daiho T. Suzuki H. Toyoshim" @default.
• W1964743049 created "2016-06-24" @default.
• W1964743049 creator A5014552722 @default.
• W1964743049 creator A5028337555 @default.
• W1964743049 creator A5072465218 @default.
• W1964743049 date "2011-08-01" @default.
• W1964743049 modified "2023-09-30" @default.
• W1964743049 title "Metal Fluoride Complexes of Na,K-ATPase" @default.
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