RESUMEN

There are four isoforms of the alpha subunit (alpha1-4) and three isoforms of the beta subunit (beta1-3) of Na,K-ATPase, with distinct tissue-specific distribution and physiological functions. alpha2 is thought to play a key role in cardiac and smooth muscle contraction and be an important target of cardiac glycosides. An alpha2-selective cardiac glycoside could provide important insights into physiological and pharmacological properties of alpha2. The isoform selectivity of a large number of cardiac glycosides has been assessed utilizing alpha1beta1, alpha2beta1, and alpha3beta1 isoforms of human Na,K-ATPase expressed in Pichia pastoris and the purified detergent-soluble isoform proteins. Binding affinities of the digitalis glycosides, digoxin, beta-methyl digoxin, and digitoxin show moderate but highly significant selectivity (up to 4-fold) for alpha2/alpha3 over alpha1 (K(D) alpha1 > alpha2 = alpha3). By contrast, ouabain shows moderate selectivity ( approximately 2.5-fold) for alpha1 over alpha2 (K(D) alpha1

Asunto(s)

Glicósidos Cardíacos/metabolismo , Glicósidos Digitálicos/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , Membrana Celular/metabolismo , Humanos , Iones , Cinética , Modelos Biológicos , Modelos Estadísticos , Miocardio/metabolismo , Pichia/metabolismo , Potasio/química , Unión Proteica , Isoformas de Proteínas , Factores de Tiempo

RESUMEN

Human alpha1 and alpha2 isoforms of Na,K-ATPase have been expressed with porcine 10*Histidine-tagged beta1 subunit in Pichia pastoris. Methanol-induced expression of alpha2 is optimal at 20 degrees C, whereas at 25 degrees C, which is optimal for expression of alpha1, alpha2 is not expressed. Detergent-soluble alpha2beta1 and alpha1beta1 complexes have been purified in a stable and functional state. alpha2beta1 shows a somewhat lower Na,K-ATPase activity and higher K0.5K compared to alpha1beta1, while values of K0.5Na and KmATP are similar. Ouabain inhibits both alpha1beta1 (K0.5 24.6 +/- 6 nM) and alpha2beta1 (K0.5 102 +/- 14 nM) with high affinity. A striking difference between the isoforms is that alpha2beta1 is unstable. Both alpha1beta1 and alpha2beta1 complexes, prepared in C12E8 with an added phosphatidyl serine, are active, but alpha2beta1 is rapidly inactivated at 0 degrees C. Addition of low concentrations of cholesterol with 1-stearoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (SOPS) stabilizes strongly, maintaining alpha2beta1 active up to two weeks at 0 degrees C. By contrast, alpha1beta1 is stable at 0 degrees C without added cholesterol. Both alpha1beta1 and alpha2beta1 complexes are stabilized by cholesterol at 37 degrees C. Human FXYD1 spontaneously associates in vitro with either alpha1beta1 or alpha2beta1, to form alpha1beta1/FXYD1 and alpha2beta1/FXYD1 complexes. The reconstituted FXYD1 protects both alpha1beta1 and alpha2beta1 very strongly against thermal inactivation. Instability of alpha2 is attributable to suboptimal phophatidylserine-protein interactions. Residues within TM8, TM9 and TM10, near the alphabeta subunit interface, may play an important role in differential interactions of lipid with alpha1 and alpha2, and affect isoform stability. Possible physiological implications of isoform interactions with phospholipids and FXYD1 are discussed.

Asunto(s)

Colesterol/farmacología , Expresión Génica , Proteínas de la Membrana/metabolismo , Fosfatidilserinas/farmacología , Fosfoproteínas/metabolismo , Pichia/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/aislamiento & purificación , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Proteínas de la Membrana/genética , Modelos Moleculares , Fosfoproteínas/genética , Pichia/genética , Unión Proteica , Desnaturalización Proteica , Estructura Cuaternaria de Proteína , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/genética , Temperatura

RESUMEN

Na+,K+-ATPase (porcine alpha1/His10*beta1 or human alpha1/porcine His10*beta1) has been expressed in Pichia pastoris and purified by Co2+-chelate affinity resin chromatography, yielding about 80% pure, functional, and stable protein in a single step. The protein was eluted in nonionic detergents together with a phosphatidylserine. Size exclusion chromatography showed that the protein eluted in n-dodecyl beta-d-maltoside is an alpha1/beta1 protomer, whereas that in octaethylene glycol dodecyl monoether contains a mixture of alpha1/beta1 protomer and higher order oligomers. The Na+,K+-ATPase activity (8-16 (mumol/min)/mg of protein) is similar in both detergents. Thus, the minimal functional unit is the alpha1/beta1 protomer, and activity is unaffected by the presence of oligomeric forms. Screening of phospholipids for stabilization of the Na+,K+-ATPase activity shows that (a) acid phospholipids are required and phosphatidylserine is somewhat better than phosphatidylinositol and (b) optimal stabilization is achieved with asymmetric phosphatidylserines having saturated (18:0 >or= 16:0) and unsaturated (18:1 > 18:2) side chains at sn-1 an sn-2 positions, respectively. In the presence of phosphatidylserine, cholesterol stabilizes the protein at 37 degrees C, but not at 0 degrees C. Cholesterol also increases the "apparent affinity" of the phosphatidylserine and stabilizes optimally in the presence of phosphatidylserines with a saturated fatty acyl chain at the sn-1 position. Ergosterol is a poor stabilizer. We propose that phosphatidylserine and cholesterol interact specifically with each other near the alpha1/beta1 subunit interface, thus stabilizing the protein. These interactions do not seem to affect Na+,K+-ATPase activity.

Asunto(s)

Membrana Celular/enzimología , Lípidos de la Membrana/metabolismo , Pichia/enzimología , ATPasa Intercambiadora de Sodio-Potasio/aislamiento & purificación , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Colesterol/farmacología , Detergentes/farmacología , Estabilidad de Enzimas/efectos de los fármacos , Expresión Génica , Humanos , Lípidos de la Membrana/química , Modelos Biológicos , Fosfolípidos/metabolismo , Pichia/genética , Pichia/ultraestructura , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/aislamiento & purificación , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/genética , Porcinos

RESUMEN

This paper provides evidence for an interaction of D443 in the N domain of Na(+),K(+)-ATPase with a Mg(2+) ion. Wild-type, D443N/A/C and S445A mutants of porcine Na(+),K(+)-ATPase (alpha1beta1) have been expressed in Pichia pastoris. By comparison with wild-type, D443N reduces the turn-over rate by about 40%. Binding affinity of ATP, measured directly, was not affected by D443N, D443A, or D443C mutations. AMP-PNP-Fe(2+)-catalyzed oxidative cleavage of Na(+),K(+)-ATPase produces two characteristic fragments, at (708)VNDS (P domain) and near (440)VAGDA (N domain), respectively. In the D443N and D443A mutants, both cleavages are suppressed, indicating an interaction between the residues with AMP-PNP-Fe(2+) bound. Previous work suggested that with ATP-Fe(2+) bound the N and P domains come into proximity, both D710 and D443 making contact with a single Fe(2+) (or Mg(2+)) ion. However, the crystal structure of Ca(2+)-ATPase with bound AMP-PCP and Mg(2+) confirm the involvement of D703 (D710) but show that E439 (D443) is too far to make contact with the Mg(2+). By contrast, in the crystal structure with bound ADP, AlF(4), and Mg(2+), representing the E(1)-P conformation, two Mg(2+) ions were observed. Significantly, ADP-Fe(2+)-mediated oxidative cleavage of renal Na,K-ATPase produces the fragment near (440)VAGDA (N domain), while the cleavage at (708)VNDS (P domain) is almost completely absent. The results are explained economically by the hypothesis that ATP is bound with two Mg(2+) (Fe(2+)) ions, a "catalytic" Mg(2+) interacting with D710 via the gamma phosphate and a "structural" Mg(2+) interacting with D443 via the alpha and beta phosphates and a water molecule, respectively.

Asunto(s)

Adenosina Trifosfato/metabolismo , Ácido Aspártico/metabolismo , Magnesio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Inhibidores Enzimáticos/metabolismo , Modelos Moleculares , Ouabaína/metabolismo , Oxidación-Reducción , Unión Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/genética , Porcinos

RESUMEN

Na+,K+-ATPase (porcine alpha/his10-beta) has been expressed in Pichia Pastoris, solubilized in n-dodecyl-beta-maltoside and purified to 70-80% purity by nickel-nitrilotriacetic acid chromatography combined with size exclusion chromatography. The recombinant protein is inactive if the purification is done without added phospholipids. The neutral phospholipid, dioleoylphosphatidylcholine, preserves Na+,K+-ATPase activity of protein prepared in a Na+-containing medium, but activity is lost in a K+-containing medium. By contrast, the acid phospholipid, dioleoylphosphatidylserine, preserves activity in either Na+- or K+-containing media. In optimal conditions activity is preserved for about 2 weeks at 0 degrees C. Both recombinant Na+,K+-ATPase and native pig kidney Na+,K+-ATPase, dissolved in n-dodecyl-beta-maltoside, appear to be mainly stable monomers (alpha/beta) as judged by size exclusion chromatography and sedimentation velocity. Na+,K+-ATPase activities at 37 degrees C of the size exclusion chromatography-purified recombinant and renal Na+,K+-ATPase are comparable but are lower than that of membrane-bound renal Na+,K+-ATPase. The beta subunit is expressed in Pichia Pastoris as two lightly glycosylated polypeptides and is quantitatively deglycosylated by endoglycosidase-H at 0 degrees C, to a single polypeptide. Deglycosylation inactivates Na+,K+-ATPase prepared with dioleoylphosphatidylcholine, whereas dioleoylphosphatidylserine protects after deglycosylation, and Na+,K+-ATPase activity is preserved. This work demonstrates an essential role of phospholipid interactions with Na+,K+-ATPase, including a direct interaction of dioleoylphosphatidylserine, and possibly another interaction of either the neutral or acid phospholipid. Additional lipid effects are likely. A role for the beta subunit in stabilizing conformations of Na+,K+-ATPase (or H+,K+-ATPase) with occluded K+ ions can also be inferred. Purified recombinant Na+,K+-ATPase could become an important experimental tool for various purposes, including, hopefully, structural work.

Asunto(s)

Ácido Nitrilotriacético/análogos & derivados , Pichia/enzimología , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/aislamiento & purificación , Adenosina Trifosfatasas/química , Animales , Bioquímica/métodos , Western Blotting , Membrana Celular/metabolismo , Cromatografía , Cromatografía Líquida de Alta Presión , Medios de Cultivo/farmacología , Electroforesis en Gel de Poliacrilamida , Vectores Genéticos , Glucósidos/química , Glicosilación , Iones , Riñón/metabolismo , Espectrometría de Masas , Ácido Nitrilotriacético/química , Compuestos Organometálicos/química , Fosfatidilcolinas/química , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Fosfolípidos/química , Plásmidos/metabolismo , Potasio/química , Unión Proteica , Conformación Proteica , Desnaturalización Proteica , Proteínas Recombinantes/química , Porcinos , Temperatura , Factores de Tiempo

RESUMEN

Na+,K+-ATPase (pig alpha1,beta1) has been expressed in the methylotrophic yeast Pichia pastoris. A protease-deficient strain was used, recombinant clones were screened for multicopy genomic integrants, and protein expression, and time and temperature of methanol induction were optimized. A 3-liter culture provides 300-500 mg of membrane protein with ouabain binding capacity of 30-50 pmol mg-1. Turnover numbers of recombinant and renal Na+,K+-ATPase are similar, as are specific chymotryptic cleavages. Wild type (WT) and a D369N mutant have been analyzed by Fe2+- and ATP-Fe2+-catalyzed oxidative cleavage, described for renal Na+,K+-ATPase. Cleavage of the D369N mutant provides strong evidence for two Fe2+ sites: site 1 composed of residues in P and A cytoplasmic domains, and site 2 near trans-membrane segments M3/M1. The D369N mutation suppresses cleavages at site 1, which appears to be a normal Mg2+ site in E2 conformations. The results suggest a possible role of the charge of Asp369 on the E1 <--> E2 conformational equilibrium. 5'-Adenylyl-beta,gamma-imidodi-phosphate(AMP-PNP)-Fe2+-catalyzed cleavage of the D369N mutant produces fragments in P (712VNDS) and N (near 440VAGDA) domains, described for WT, but only at high AMP-PNP-Fe2+ concentrations, and a new fragment in the P domain (near 367CSDKTGT) resulting from cleavage. Thus, the mutation distorts the active site. A molecular dynamic simulation of ATP-Mg2+ binding to WT and D351N structures of Ca2+-ATPase (analogous to Asp369 of Na+,K+-ATPase) supplies possible explanations for the new cleavage and for a high ATP affinity, which was observed previously for the mutant. The Asn351 structure with bound ATP-Mg2+ may resemble the transition state of the WT poised for phosphorylation.

Asunto(s)

Pichia/enzimología , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , ATPasa Intercambiadora de Sodio-Potasio/química , Animales , Ácido Aspártico/química , Sitios de Unión , Western Blotting , Membrana Celular/metabolismo , Quimotripsina/farmacología , Citoplasma/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Vectores Genéticos , Genoma Fúngico , Hierro/química , Magnesio/química , Modelos Moleculares , Mutación , Oxígeno/metabolismo , Fosforilación , Plásmidos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Porcinos , Temperatura , Factores de Tiempo

Asunto(s)

Hierro/farmacología , Pichia/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Catálisis , Cinética , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , Porcinos

RESUMEN

CHIF (corticosteroid hormone-induced factor) is a member of the FXYD family that shares approximately 50% homology with the gamma subunit of Na,K-ATPase. It is expressed in renal collecting duct and distal colon, and is upregulated by Na(+) deprivation and high K(+) diet. Both CHIF and gamma are coimmunoprecipitated by an anti-alpha subunit antibody, and alpha is immunoprecipitated by anti-gamma and anti-CHIF antibodies. (86)Rb(+) flux experiments in CHIF-transfected HeLa cells demonstrate that CHIF increases the affinity for cytoplasmic Na(+), but does not affect the affinity for extracellular K(Rb). A physiological role of CHIF in kidney function is further elucidated by the phenotypic analysis of CHIF knockout mice. Taken together with data by others, it appears that FXYD proteins are tissue-specific subunits or regulators of the Na,K-ATPase whose function is to adjust the pump kinetics to particular physiological needs.

Asunto(s)

Proteínas de la Membrana/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Secuencia de Aminoácidos , Células HeLa , Homeostasis , Humanos , Bombas Iónicas/metabolismo , Proteínas de la Membrana/química , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rubidio/metabolismo , Homología de Secuencia de Aminoácido , Transfección

Asunto(s)

Proteínas de Unión al Calcio/metabolismo , Proteínas de la Membrana/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Sitios de Unión , Proteínas de Unión al Calcio/química , Bovinos , Membrana Celular/enzimología , Colon , Mucosa Intestinal/enzimología , Médula Renal/enzimología , Proteínas de la Membrana/química , Ratas , Sarcolema/enzimología

RESUMEN

This work utilizes Fe(2+)-catalyzed cleavages and molecular modeling to obtain insight into conformations of cytoplasmic domains and ATP-Mg(2+) binding sites of Na(+),K(+)-ATPase. In E(1) conformations the ATP-Fe(2+) complex mediates specific cleavages at 712VNDS (P domain) and near 440VAGDA (N domain). In E(2)(K), ATP-Fe(2+) mediates cleavages near 212TGES (A domain), near 440VAGDA, and between residues 460-490 (N domain). Cleavages at high ATP-Fe(2+) concentrations do not support suggestions for two ATP sites. A new reagent, fluorescein-DTPA, has been synthesized. The fluorescein-DTPA-Fe(2+) complex mediates cleavages similar to those mediated by ATP-Fe(2+). The data suggest the existence of N to P domain interactions in E(1)Na, with bound ATP-Fe(2+) or fluorescein-DPTA-Fe(2+), A-N, and A-P interactions in E(2)(K), and provide testable constraints for model building. Molecular models based on the Ca(2+)-ATPase structure are consistent with the predictions. Specifically, high-affinity ATP-Mg(2+) binding in E(1) is explained with the N domain tilted ca. 80 degrees toward the P domain, by comparison with well-separated N and P domains in the Ca-ATPase crystal structure. With ATP-Mg(2+) docked, bound Mg(2+) is close to both D710 (in 710DGVNDS) and D443 (in 440VAGDASE). D710 is known to be crucial for Mg(2+) binding. The cleavage and modeling data imply that D443 could also be a candidate for Mg(2+) binding. Comparison of E(1).ATP,Mg(2+) and E(2) models suggests an explanation of the high or low ATP affinities, respectively. We propose a scheme of ATP-Mg(2+) and Mg(2+) binding and N, P, and A domain interactions in the different conformations of the catalytic cycle.

Asunto(s)

Adenosina Trifosfato/química , Simulación por Computador , Citoplasma/enzimología , Compuestos Ferrosos/química , Modelos Moleculares , ATPasa Intercambiadora de Sodio-Potasio/química , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Catálisis , Compuestos Ferrosos/metabolismo , Fluoresceína/química , Hidrólisis , Oxidación-Reducción , Ácido Pentético/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Porcinos

RESUMEN

Like the gamma-subunit of Na-K-ATPase, the corticosteroid hormone-induced factor (CHIF) is a member of the FXYD family of one-transmembrane-segment proteins. Both CHIF and two splice variants of gamma, gamma(a) and gamma(b), are expressed in the kidney. Immunolocalization experiments demonstrate mutually exclusive expression of CHIF and gamma in different nephron segments. Specific coimmunoprecipitation experiments demonstrate the existence in kidney membranes of the complexes alpha/beta/gamma(a), alpha/beta/gamma(b), and alpha/beta/CHIF and exclude mixed complexes such as alpha/beta/gamma(a)/gamma(b) and alpha/beta/gamma/CHIF. CHIF has been expressed in HeLa cells harboring the rat alpha(1)-subunit of Na-K-ATPase. (86)Rb flux experiments demonstrate that CHIF induces a two- to threefold increase in apparent affinity for cytoplasmic Na (K'(Na)) but does not affect affinity for extracellular K (Rb) ions (K'(K)) or V(max). Measurements of Na-K-ATPase using isolated membranes show similar but smaller effects of CHIF on K'(Na), whereas K'(K) and K'(ATP) are unaffected. The functional effects of CHIF differ from those of gamma. An implication of these findings is that other FXYD proteins could act as tissue-specific modulators of Na-K-ATPase.

Asunto(s)

Proteínas de la Membrana/fisiología , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Animales , Antibacterianos/farmacología , Colon/enzimología , Colon/metabolismo , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Higromicina B/farmacología , Inmunohistoquímica , Riñón/enzimología , Riñón/metabolismo , Cinética , Ouabaína/farmacología , Pruebas de Precipitina , Ratas , Radioisótopos de Rubidio , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Transfección