Cys577 Is a Conformationally Mobile Residue in the ATP-binding Domain of the Na,K-ATPase α-Subunit

2-[4′-Maleimidylanilino]naphthalene 6-sulfonic acid (MIANS) irreversibly inactivates Na,K-ATPase in a time- and concentration-dependent manner. Inactivation is prevented by 3 mm ATP or low K+ (<1 mm); the protective effect K+ is reversed at higher concentrations. This biphasic effect was also observed with K+ congeners. In contrast, Na+ ions did not protect. MIANS inactivation disrupted high affinity ATP binding. Tryptic fragments of MIANS-labeled protein were analyzed by reversed phase high performance liquid chromatography. ATP clearly protected one major labeled peptide peak. This observation was confirmed by separation of tryptic peptides in SDS-polyacrylamide gel electrophoresis revealing a single fluorescently-labeled peptide of ∼5 kDa. N-terminal amino acid sequencing identified the peptide (V545LGFCH …). This hydrophobic peptide contains only two Cys residues in all sodium pump α-subunit sequences and is found in the major cytoplasmic loop between M4 and M5, a region previously associated with ATP binding. Subsequent digestion of the tryptic peptide with V8 protease and N-terminal amino acid sequencing identified the modified residue as Cys577. The cation-dependent change in reactivity of Cys577implies structural alterations in the ATP-binding domain following cation binding and occlusion in the intramembrane domain of Na,K-ATPase and expands our knowledge of the extent to which cation binding and occlusion are sensed in the ATP hydrolysis domain. 2-[4′-Maleimidylanilino]naphthalene 6-sulfonic acid (MIANS) irreversibly inactivates Na,K-ATPase in a time- and concentration-dependent manner. Inactivation is prevented by 3 mm ATP or low K+ (<1 mm); the protective effect K+ is reversed at higher concentrations. This biphasic effect was also observed with K+ congeners. In contrast, Na+ ions did not protect. MIANS inactivation disrupted high affinity ATP binding. Tryptic fragments of MIANS-labeled protein were analyzed by reversed phase high performance liquid chromatography. ATP clearly protected one major labeled peptide peak. This observation was confirmed by separation of tryptic peptides in SDS-polyacrylamide gel electrophoresis revealing a single fluorescently-labeled peptide of ∼5 kDa. N-terminal amino acid sequencing identified the peptide (V545LGFCH …). This hydrophobic peptide contains only two Cys residues in all sodium pump α-subunit sequences and is found in the major cytoplasmic loop between M4 and M5, a region previously associated with ATP binding. Subsequent digestion of the tryptic peptide with V8 protease and N-terminal amino acid sequencing identified the modified residue as Cys577. The cation-dependent change in reactivity of Cys577implies structural alterations in the ATP-binding domain following cation binding and occlusion in the intramembrane domain of Na,K-ATPase and expands our knowledge of the extent to which cation binding and occlusion are sensed in the ATP hydrolysis domain. polyacrylamide gel electrophoresis high performance liquid chromatography 2-(4′-maleimidylanilino)naphthalene 6-sulfonic acid N-Tris-(hydroxymethyl)-methylglycine 3-(cyclohexylamino)propanesulfonic acid N, N, N′, N′-tetramethylethylenediamine diisothiocyanatostilbene-2,2′-disulfonate The Na,K-ATPase (EC 3.6.1.37) is a P2-type ATPase (1Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (416) Google Scholar) that is responsible for the maintenance of the sodium and potassium ion gradients in most eukaryotic cells. The integral membrane protein is a heterodimeric enzyme comprised of an α-subunit of about 100 kDa and a β-subunit, which migrates in SDS-PAGE1 with an apparent mass of 55 kDa. The α-subunit is composed of 10 putative transmembrane segments, several of which are thought to form an intramembrane cation-binding domain and a major cytoplasmic loop between the fourth and fifth transmembrane segments (2Lingrel J.B Kuntzweiler T. J. Biol. Chem. 1994; 269: 19659-19662Abstract Full Text PDF PubMed Google Scholar). This intracellular loop of around 45 kDa contains the catalytic phosphorylation site (Asp369) and the ATP-binding domain (for review see Ref. 2Lingrel J.B Kuntzweiler T. J. Biol. Chem. 1994; 269: 19659-19662Abstract Full Text PDF PubMed Google Scholar). A variety of reagents that have been used to inactivate the Na,K-ATPase in an ATP-protectable fashion have identified several putative contact residues in the ATP-binding domain. These have been predominantly lysine-reactive reagents, and a series of residues have been identified (3Kaplan J.H. Kaplan J.H. De Weer P. The Sodium Pump: Structure, Mechanism and Regulation. Rockefeller University, New York1991: 117-128Google Scholar). It has been reported that the reactivity of several of these residues is dependent upon cation binding and thus enzyme conformation (4Ellis-Davies G.C.R. Kaplan J.H. J. Biol. Chem. 1990; 265: 20570-20576Abstract Full Text PDF PubMed Google Scholar, 5Gatto C. Lutsenko S. Kaplan J.H. Arch. Biochem. Biophys. 1997; 340: 90-100Crossref PubMed Scopus (34) Google Scholar, 6Kaplan J.H. Gatto C. Holden J.P. Thornewell S.J. Acta Scand. Physiol. 1997; 643: 99-105Google Scholar). The dual function of ATP hydrolysis and cation transport, which constitute ion pumping, mechanistically involve the coupled interactions of the ATP-binding domain and the cation-binding domain. Regions of the α-subunit and some specific amino acid residues have been identified as being close to (or at) the ATP-binding region of the enzyme. To date, all of the residues identified lie in the major cytoplasmic loop of ∼440 amino acids, between transmembrane helices 4 and 5. Recent evidence that bacterial overexpression of the M4M5 loop yields a peptide with the same nucleoside phosphate binding specificity as the intact Na,K-ATPase lends further support to the notion that this segment supplies the ATP-binding residues in the intact sodium pump (7Gatto C. Wang A.X. Kaplan J.H. J. Biol. Chem. 1998; 273: 10578-10585Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The majority of the amino acids in the ATP-binding domain identified by direct labeling have been lysine residues, primarily because of the greater availability of lysine-selective reagents. These residues include Lys480, Lys487, Lys501, Lys589, Lys605, Lys618, Lys622, and Lys719 (for review see Refs. 2Lingrel J.B Kuntzweiler T. J. Biol. Chem. 1994; 269: 19659-19662Abstract Full Text PDF PubMed Google Scholar and 3Kaplan J.H. Kaplan J.H. De Weer P. The Sodium Pump: Structure, Mechanism and Regulation. Rockefeller University, New York1991: 117-128Google Scholar). In addition, cysteine-directed reagents have been used in a variety of transport systems, e.g. P-glycoprotein (8Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 9Lui R. Sharom F.J. Biochemistry. 1996; 35: 11865-11873Crossref PubMed Scopus (191) Google Scholar) and lac permease (10Jung H. Jung K. Kaback H.R. Biochemistry. 1994; 33: 12160-12165Crossref PubMed Scopus (53) Google Scholar). Such studies in the gastric H,K-ATPase (11Besancon M. Simon A. Sachs G. Shin J.M. J. Biol. Chem. 1997; 272: 22438-22446Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar) led to the identification of important extracytosolic cysteine residues unique to this member of the P-type ATPase family. The early use of cysteine-directed reagents in the study of Na,K-ATPase also led to important insights. The consequences of treating the enzyme with N-ethylmaleimide led to inhibition of the major phosphoenzyme transition (E1P E2P) and resulted in the formulation of the widely accepted Post-Albers scheme for the reaction pathway (12Albers R.W. Annu. Rev. Biochem. 1967; 36: 727-756Crossref PubMed Scopus (578) Google Scholar, 13Post R.L. Hegyvary C. Kume S. J. Biol. Chem. 1972; 247: 6530-6540Abstract Full Text PDF PubMed Google Scholar). A previous kinetic study of the interactions of MIANS with the Na,K-ATPase reported effects of ATP in modulating the resulting inactivation of the enzyme (14Gupta S.S. Lane L.K. J. Biol. Chem. 1979; 254: 10362-10367Abstract Full Text PDF PubMed Google Scholar). More recently, studies using fluorescent cysteine reagents on a sided preparation of Na,K-ATPase demonstrated that only two Cys residues (of the possible seven intramembrane cysteines) were exposed to the extracellular medium (15Lutsenko S. S. Kaplan J.H. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). These the of a to the membrane of the Na,K-ATPase α-subunit Kaplan J.H. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar). In the evidence that MIANS inactivates Na,K-ATPase by to which in the of high affinity ATP binding. also that the reactivity of is and that reactivity in This that structural in the ATP-binding domain are at 100 amino acid residues in the and that the binding of of the two K+ ions in the ATP-binding site and acid were and high and low were was low and were and sodium phosphate were potassium and were membrane was MIANS was were acid and were and sodium sodium sodium and were was Na,K-ATPase was as by Biophys. PubMed Scopus Google Scholar) with the that enzyme was a with a The enzyme was greater SDS-polyacrylamide gel was by the of J. Biol. Chem. Full Text PDF PubMed Google Scholar) using as a Na,K-ATPase was in a medium mm mm mm 3 3 mm mm and enzyme. The was at for and the of enzyme ATP hydrolysis was as by Biochem. Biophys. PubMed Scopus Google Scholar). The enzyme used in these studies a specific of The enzyme was in mm MIANS in was at the and the reaction was at The reaction was by with mm mm mm was the reaction and ATPase was as The of was in the reaction and in the with were for to MIANS Na,K-ATPase was for 10 at in the with 10 mm mm 3 mm or 3 mm and MIANS was to a that resulted in at inactivation the of and the reaction was at for The reaction was by with was the reaction for an ATPase and the protein was in a at at 4 for The was in to a protein of A of this protein was with and mm was to PubMed Scopus Google Scholar) in Na,K-ATPase α-subunit labeled with MIANS was by on with a protein were observed by the gel with R. of enzyme was labeled with MIANS in the and of 3 mm for the was for an ATPase and the protein was at at 4 for The was in was and the was at for The digestion was by the of and the was the membrane by at MIANS-labeled α-subunit was a gel and the gel with mm for a of at The protein was with a and with of protein was the membrane with and the of protein was by the of J. Biol. Chem. Full Text PDF PubMed Google Scholar). digestion was in mm with V8 protease for at fragments were with 10 of for peptide fragments digestion were by using a phase The was with a the pump A and pump The was and the was with a were in and the peptide fragments of were with a The was to with and further on a with the same and to with as peptide fragments were with of at for peptide fragments were in and one of was to the These membrane were by the of 10 of at for The peptides were at and in were on a gel to and H. G. Biochem. PubMed Scopus Google Scholar). electrophoresis protein fragments were to membrane by in 10 mm P. J. Biol. Chem. Full Text PDF PubMed Google Scholar) at for labeled were for N-terminal amino acid sequencing and amino acids University, binding was to the of J. Biochem. PubMed Scopus Google Scholar) with in a medium mm mm mm and enzyme. Na,K-ATPase was labeled with MIANS in the of 10 mm or 3 mm A reaction was Na,K-ATPase was as The labeled enzyme was two and the protein was at for at 4 was in of A mm mm and the was in A with 10 mm The were at for and on for was and the were on for was at for and the was A was to the and was in of The of to the enzyme was in a with The of protein was in by the of using as a of enzyme with MIANS at resulted in a in the Na,K-ATPase that was dependent on the of MIANS and the of at The of MIANS inactivation is not a single that one of cysteine residues are with previous (14Gupta S.S. Lane L.K. J. Biol. Chem. 1979; 254: 10362-10367Abstract Full Text PDF PubMed Google Scholar). that the inactivation reaction is at a single site and that to MIANS in labeling of several 3 and labeling were and MIANS to the specific site of inactivation of the effect of cation on Na,K-ATPase inactivation by Na,K-ATPase was at for with MIANS in the of of enzyme are at 55 mm for not to a with between and mm the of and the the are not lie the of Na,K-ATPase labeled with MIANS in the of MIANS-labeled protein was by on a polyacrylamide The MIANS-labeled were by with a the α-subunit was labeled in all MIANS was Lane mm mm 10 mm mm of tryptic peptides MIANS-labeled Na,K-ATPase reversed phase Na,K-ATPase was labeled with MIANS in the and of ATP at all peptide fragments and at MIANS-labeled peptide The is of tryptic peptides MIANS-labeled Na,K-ATPase reversed phase Na,K-ATPase was labeled with MIANS in the and of ATP at all peptide fragments and at MIANS-labeled peptide The is The of enzyme in the reaction the extent of Na,K-ATPase inactivation It that Na+ ions the of ATP or K+ ions protected the enzyme inactivation The of and phosphate ions did not the enzyme These that inactivation of Na,K-ATPase by MIANS is dependent upon the enzyme or or and the sodium pump protein the in A was to SDS-polyacrylamide gel and MIANS labeling was The was not labeled by The of α-subunit labeled in the of was to the of enzyme inactivation was observed in the reaction MIANS In the of ATP and to a extent the of labeling by MIANS was as with enzyme labeled in the of or in the of Na+ or The in A that Na+ and K+ ions have effects on the enzyme reactivity This to in with previous that all and the reaction of MIANS with the Na,K-ATPase (14Gupta S.S. Lane L.K. J. Biol. Chem. 1979; 254: 10362-10367Abstract Full Text PDF PubMed Google Scholar). and Lane (14Gupta S.S. Lane L.K. J. Biol. Chem. 1979; 254: 10362-10367Abstract Full Text PDF PubMed Google Scholar) the MIANS associated with enzyme and not enzyme inactivation as in our all of MIANS that to the our only that in enzyme is that a low of binding inactivation 3 It is that these are the for the of the effects of on MIANS The protective effect with 10 mm K+ to between MIANS inactivation in the of of several These clearly that the of Na+ ions enzyme inactivation by at low (<1 or high In contrast, low of K+ (<1 or protected MIANS at higher of this effect was reversed and enzyme inactivation was not This biphasic effect was previously observed with C. Lutsenko S. Kaplan J.H. Arch. Biochem. Biophys. 1997; 340: 90-100Crossref PubMed Scopus (34) Google Scholar, Kaplan J.H. Biochemistry. PubMed Scopus Google Kaplan J.H. PubMed Scopus Google and J.H. Gatto C. Holden J.P. Thornewell S.J. Acta Scand. Physiol. 1997; 643: 99-105Google that also inactivate Na,K-ATPase in an ATP-protectable and of have been to the ATP-binding loop It is that the with MIANS labeling in the of 10 mm K+ was because this between that that It is that the between K+ with and labeling at J.H. Gatto C. Holden J.P. Thornewell S.J. Acta Scand. Physiol. 1997; 643: 99-105Google Kaplan J.H. Biochemistry. PubMed Scopus Google Scholar) were the K+ MIANS 3 The between the cation-binding the for this to A for this a high of the 3 the extent of MIANS the α-subunit in the of The of MIANS the biphasic with is some MIANS the α-subunit in which enzyme is that some cysteine residues are modified by This labeling that is not associated with inactivation the between a previous study (14Gupta S.S. Lane L.K. J. Biol. Chem. 1979; 254: 10362-10367Abstract Full Text PDF PubMed Google Scholar) and this of sodium pump by MIANS resulted in a of Na,K-ATPase that was prevented by with 3 mm A at the of ATP MIANS inactivation that of sodium pump protein with of ATP greater resulted in the same of observed with 3 mm in previous The apparent for ATP was ATP binding is MIANS and because the of the low affinity ATP site in the of K+ is is to with this ATP effect as the high or low affinity binding to or ATP inactivation that MIANS to a residue in the ATP To further this high affinity binding Na,K-ATPase was labeled with MIANS in the and of 3 mm ATP or in the of 10 mm of enzyme were used to Na,K-ATPase and binding The enzyme and binding were in the enzyme not enzyme protected MIANS inactivation The of of protein to to the of enzyme that the of high affinity ATP with the of It to that the of ATPase is a of the of high affinity binding that is for enzyme phosphorylation and ATPase Na,K-ATPase was labeled with MIANS in the and of 3 mm ATP and with The tryptic fragments were the fragments and all of the MIANS labeling was associated with the of the tryptic peptides was by phase on a using with acid by a acid to of and at and are for sodium pump labeled in the and of 3 mm The of tryptic peptides at were that the digestion and chromatography were and not by MIANS The at MIANS-labeled several peptides the MIANS the peptide enzyme labeled in the or of ATP by a single MIANS clearly at in the A and of the two at also the of this in the The the ATP-protectable was to separation of the MIANS-labeled The of the was to with and a to was at The MIANS-labeled peptide as a single at not to the MIANS-labeled tryptic was separation polyacrylamide gel electrophoresis on a gel H. G. Biochem. PubMed Scopus Google Scholar). was the with the MIANS was associated with the peptide a single fluorescent was observed peptides labeled in that of ATP the peptides the protected by ATP an with are for cysteine The MIANS-labeled which was identified the gel was for N-terminal amino acid several a hydrophobic peptide at The peptide that by at and This peptide is 45 amino acid residues in and in most Na,K-ATPase contains two cysteine residues, and as for MIANS The protein reported for Na,K-ATPase α-subunit is the a Ref. H. Wang A. R.W. The Sodium Pump: Mechanism and in Google Scholar). The amino acid in the region of the MIANS-labeled peptide all Na,K-ATPase a cysteine residue at (or analyzed using Ref. J. 1997; PubMed Scopus Google Scholar). the reported is for our residue the reported is in and residue is in cysteine and not our or Cys577. this to the because in all of our sequencing a amino acid in the fifth the in the fourth and were The that amino acid sequencing of the MIANS-labeled peptide did not at is an that the protein of the is and all contains a Cys at this MIANS was to or because cysteine residues are not identified in N-terminal amino acid MIANS-labeled Na,K-ATPase α-subunit with protease V8 that because were residues between and Cys577. V8 the peptide fragments were on a and a single fluorescent was observed and for The peptide was identified as that MIANS was to Cys577. In this have evidence that in the α-subunit of Na,K-ATPase is modified by of this cysteine in inactivation of the enzyme and of ATP binding. The of ATP with MIANS inactivation and labeling of the enzyme. These evidence for the of in the ATP-binding of In addition, the reactivity of this residue are about the of the cation The cytoplasmic loop of the Na,K-ATPase α-subunit has been associated with ATP binding and phosphorylation of the pump the reaction specific amino acids to in the ATP-binding have been identified by studies (for review see Ref. 2Lingrel J.B Kuntzweiler T. J. Biol. Chem. 1994; 269: 19659-19662Abstract Full Text PDF PubMed Google Scholar). this is the is not close in the to of the previously identified amino acids Lys501, Lys480, and and a region of the M4M5 loop in ATP binding. This peptide has previously been thought to associated with the because of hydrophobic and as a of studies with reagents. this peptide was labeled with hydrophobic reagents as H. J. Biol. Chem. Full Text PDF PubMed Google and Biochemistry. PubMed Scopus Google Scholar). in contrast, this peptide has been identified in the of the membrane preparation of Na,K-ATPase to hydrolysis PubMed Scopus Google Scholar). A study of the of digestion of the Na,K-ATPase that this peptide associated with the membrane or in the upon which were digestion S. Kaplan J.H. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). Such evidence that this peptide is in the membrane or on the of the intramembrane of It is also possible that this peptide form a hydrophobic of binding reagents. sequences P-type have been with the of the pump the structural J. Biochem. PubMed Scopus Google Scholar, Acta Physiol. Scand. Scholar, The Sodium Pump: Mechanism and in New Google Scholar). These are with the of the of the in the ATP-binding domain. A of Na,K-ATPase with the structural that the hydrophobic peptide labeled by MIANS is by and J. Biochem. PubMed Scopus Google Scholar). In this lie at the of and in the of The this in close to residues as and Lys480, of which has been to in ATP-protectable enzyme inactivation Acta Physiol. Scand. Scholar). this the of the MIANS-labeled peptide and the ATP-binding Acta Physiol. Scand. Scholar). The is P-type and evidence studies of the Na,K-ATPase that this region is the ion site M. Wang K. Biochemistry. 1997; 36: PubMed Scopus Google Scholar, M. Biochemistry. 1997; 36: PubMed Scopus Google Scholar). It is further that this ion the enzyme by the and of ATP and in to is M. Biochemistry. 1997; 36: PubMed Scopus Google Scholar). In the of this one the of a as MIANS at the enzyme and with ATP MIANS The observation that the for inactivation by is in the loop and protected reaction with MIANS by the of ATP that this residue is in the ATP-binding domain. In addition, the inactivation of the enzyme that is to a of high affinity ATP binding is not is a contact site for ATP binding in the domain or MIANS an effect by of or by of the of this region following It is that to these only the or the cytoplasmic loop the ATP-binding is in the of Recent studies that this a and a peptide to the M4M5 loop has been to ATP-protectable labeling by MIANS (7Gatto C. Wang A.X. Kaplan J.H. J. Biol. Chem. 1998; 273: 10578-10585Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). is also evidence that is in the ATP-binding domain. has been reported previously that several which the loop of the the enzyme is in the form in the These include J.H. Gatto C. Holden J.P. Thornewell S.J. Acta Scand. Physiol. 1997; 643: 99-105Google (4Ellis-Davies G.C.R. Kaplan J.H. J. Biol. Chem. 1990; 265: 20570-20576Abstract Full Text PDF PubMed Google and C. Lutsenko S. Kaplan J.H. Arch. Biochem. Biophys. 1997; 340: 90-100Crossref PubMed Scopus (34) Google Kaplan J.H. Biochemistry. PubMed Scopus Google Scholar). This in reactivity upon sodium binding is of the cation-binding region of the to the intramembrane segments, the of the ATP-binding domain in the all of these with Lys501, was these the thus to the or were and to the region close to In the found that also the reactivity of that cation binding effects are not to are a the It is that been to close to in the MIANS of a biphasic on K+ ion at low the of K+ ions at high K+ ion the effect is reversed and A observation was reported for inactivation of the Na,K-ATPase Kaplan J.H. Biochemistry. PubMed Scopus Google Scholar) and with several with J.H. Gatto C. Holden J.P. Thornewell S.J. Acta Scand. Physiol. 1997; 643: 99-105Google Scholar). It that this biphasic effect has to the of the pump as the by ATP site and was in the of K+ as and and Refs. 5Gatto C. Lutsenko S. Kaplan J.H. Arch. Biochem. Biophys. 1997; 340: 90-100Crossref PubMed Scopus (34) Google Scholar and 6Kaplan J.H. Gatto C. Holden J.P. Thornewell S.J. Acta Scand. Physiol. 1997; 643: 99-105Google Scholar). several to this the is that the enzyme conformation with a single K+ ion that with two K+ ions (for see Ref. 6Kaplan J.H. Gatto C. Holden J.P. Thornewell S.J. Acta Scand. Physiol. 1997; 643: 99-105Google Scholar). This in the and or a change in the ATP site is and reactivity MIANS this on the Na,K-ATPase with MIANS on the of reaction of Cys residues the modified residues which inactivation (14Gupta S.S. Lane L.K. J. Biol. Chem. 1979; 254: 10362-10367Abstract Full Text PDF PubMed Google Scholar). These did not effects in the labeling effects of which the site and extent of reaction of protein and Ref. S.S. Lane L.K. J. Biol. Chem. 1979; 254: 10362-10367Abstract Full Text PDF PubMed Google Scholar and this The transport of Na+ and K+ ions the hydrolysis of ATP to the transmembrane of these These coupled to at of the To the of cation transport, is to these protein with Recent studies or of the Na,K-ATPase has identified the region as being in cation binding and occlusion J.M. Kaplan J.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.M. Kaplan J.H. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, J.M. J.B J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). In are residues, J.M. Kaplan J.H. J. Biol. Chem. Full Text PDF PubMed Google J.M. Kaplan J.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.M. Kaplan J.H. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, Scholar, J.M. J. J.B J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google and J.B Biochemistry. PubMed Scopus Google that to in cation that this the catalytic and an in the cation in Na,K-ATPase S. R. Kaplan J.H. S. A. 1995; PubMed Scopus Google Scholar) and the H,K-ATPase C. Lutsenko S. Shin J.M. Sachs G. Kaplan J.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The that with the extracellular loop between and is with inhibition and of binding M. Kuntzweiler J.M. J.B J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). In a to the identification of the in cation studies and of the loop of the α-subunit have that most of the ATP-binding residues are in the cytoplasmic loop between M4 and (7Gatto C. Wang A.X. Kaplan J.H. J. Biol. Chem. 1998; 273: 10578-10585Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, Kaplan J.H. J. Physiol. 1990; PubMed Google Scholar). The in this residue that to in a with ATP binding. the that the around K+ ions or Na+ ions are in the cation-binding domain. evidence has also that the of several residues or segments with to the change as the enzyme S. Kaplan J.H. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). It is that these structural are the for the ATP affinity that the enzyme Na+ for K+ at binding R.L. Hegyvary C. Kume S. J. Biol. Chem. 1972; 247: 6530-6540Abstract Full Text PDF PubMed Google Scholar). that with as MIANS to the of the ATP-binding domain that and for are also to R. University, for a about the α-subunit