Phospholemman (FXYD1) Raises the Affinity of the Human α1β1 Isoform of Na,K-ATPase for Na Ions

Lade...
Vorschaubild
Dateien
apell_phospholemman.pdf
apell_phospholemman.pdfGröße: 10.37 MBDownloads: 754
Datum
2011
Autor:innen
Katz, Adriana
Mishra, Neeraj Kumar
Belogus, Talya
Lifshitz, Yael
Garty, Haim
Karlish, Steven J. D.
Herausgeber:innen
Kontakt
ISSN der Zeitschrift
Electronic ISSN
ISBN
Bibliografische Daten
Verlag
Schriftenreihe
Auflagebezeichnung
DOI (zitierfähiger Link)
ArXiv-ID
Internationale Patentnummer
Angaben zur Forschungsförderung
Projekt
Open Access-Veröffentlichung
Open Access Green
Sammlungen
Core Facility der Universität Konstanz
Gesperrt bis
Titel in einer weiteren Sprache
Forschungsvorhaben
Organisationseinheiten
Zeitschriftenheft
Publikationstyp
Zeitschriftenartikel
Publikationsstatus
Published
Erschienen in
Zusammenfassung

The human α1/His10-β1 isoform of the Na,K-ATPase has been expressed in Pichia pastoris, solubilized in n-dodecyl-β-maltoside and purified by metal chelate chromatography. The α1β1 complex spontaneously associates in vitro with the detergent-solubilized purified human FXYD1 (phospholemman) expressed in Escherichia coli. It has been confirmed that FXYD1 spontaneously associates in vitro with the α1/His10-β1 complex and stabilizes it in an active mode. The functional properties of the α1/His10-β1 and α1/His10-β1/FXYD1 complexes have been investigated by fluorescence methods. The electrochromic dye RH421 which monitors binding to and release of ions from the binding sites has been applied in equilibrium titration experiments to determine ion binding affinities and revealed that FXYD1 induces an ~30% increase of the Na+-binding affinity in both the E1 and P-E2 conformations. By contrast, it does not affect the affinities for K+ and Rb+ ions. Phosphorylation induced partial reactions of the enzyme have been studied as backdoor phosphorylation by inorganic phosphate and in kinetic experiments with caged ATP in order to evaluate the ATP-binding affinity and the time constant of the conformational transition, Na3E1-P → P-E2Na3. No significant differences with or without FXYD1 could be detected. Rate constants of the conformational transitions Rb2E1→ E2(Rb2) and E2(Rb2) → Na3E1, investigated with fluorescein-labeled Na,K-ATPase showed only minor or no effects of FXYD1, respectively. The conclusion from all these experiment is that FXYD1 raises the binding affinity of α1β1 for Na ions, presumably at the third Na-selective binding site. In whole cell expression studies FXYD1 reduces the apparent affinity for Na ions. Possible reasons for the difference from this study using the purified recombinant Na,K-ATPase are discussed.

Zusammenfassung in einer weiteren Sprache
Fachgebiet (DDC)
570 Biowissenschaften, Biologie
Schlagwörter
Konferenz
Rezension
undefined / . - undefined, undefined
Zitieren
ISO 690CIRRI, Erica, Adriana KATZ, Neeraj Kumar MISHRA, Talya BELOGUS, Yael LIFSHITZ, Haim GARTY, Steven J. D. KARLISH, Hans-Jürgen APELL, 2011. Phospholemman (FXYD1) Raises the Affinity of the Human α1β1 Isoform of Na,K-ATPase for Na Ions. In: Biochemistry. 2011, 50(18), pp. 3736-3748. ISSN 0006-2960. eISSN 1520-4995. Available under: doi: 10.1021/bi2001714
BibTex
@article{Cirri2011-05-10Phosp-14178,
  year={2011},
  doi={10.1021/bi2001714},
  title={Phospholemman (FXYD1) Raises the Affinity of the Human α1β1 Isoform of Na,K-ATPase for Na Ions},
  number={18},
  volume={50},
  issn={0006-2960},
  journal={Biochemistry},
  pages={3736--3748},
  author={Cirri, Erica and Katz, Adriana and Mishra, Neeraj Kumar and Belogus, Talya and Lifshitz, Yael and Garty, Haim and Karlish, Steven J. D. and Apell, Hans-Jürgen}
}
RDF
<rdf:RDF
    xmlns:dcterms="http://purl.org/dc/terms/"
    xmlns:dc="http://purl.org/dc/elements/1.1/"
    xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#"
    xmlns:bibo="http://purl.org/ontology/bibo/"
    xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#"
    xmlns:foaf="http://xmlns.com/foaf/0.1/"
    xmlns:void="http://rdfs.org/ns/void#"
    xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > 
  <rdf:Description rdf:about="https://kops.uni-konstanz.de/server/rdf/resource/123456789/14178">
    <dc:creator>Mishra, Neeraj Kumar</dc:creator>
    <dc:contributor>Apell, Hans-Jürgen</dc:contributor>
    <dcterms:title>Phospholemman (FXYD1) Raises the Affinity of the Human α1β1 Isoform of Na,K-ATPase for Na Ions</dcterms:title>
    <dc:creator>Apell, Hans-Jürgen</dc:creator>
    <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2012-02-01T10:20:51Z</dc:date>
    <dcterms:abstract xml:lang="eng">The human α1/His10-β1 isoform of the Na,K-ATPase has been expressed in Pichia pastoris, solubilized in n-dodecyl-β-maltoside and purified by metal chelate chromatography. The  α1β1 complex spontaneously associates in vitro with the detergent-solubilized purified human FXYD1 (phospholemman) expressed in Escherichia coli. It has been confirmed that FXYD1 spontaneously associates in vitro with the α1/His10-β1 complex and stabilizes it in an active mode. The functional properties of the α1/His10-β1 and α1/His10-β1/FXYD1 complexes have been investigated by fluorescence methods. The electrochromic dye RH421 which monitors binding to and release of ions from the binding sites has been applied in equilibrium titration experiments to determine ion binding affinities and revealed that FXYD1 induces an ~30% increase of the Na+-binding affinity in both the E1 and P-E2 conformations. By contrast, it does not affect the affinities for K+ and Rb+ ions. Phosphorylation induced partial reactions of the enzyme have been studied as backdoor phosphorylation by inorganic phosphate and in kinetic experiments with caged ATP in order to evaluate the ATP-binding affinity and the time constant of the conformational transition, Na3E1-P → P-E2Na3. No significant differences with or without FXYD1 could be detected. Rate constants of the conformational transitions Rb2E1→ E2(Rb2) and E2(Rb2) → Na3E1, investigated with fluorescein-labeled Na,K-ATPase showed only minor or no effects of FXYD1, respectively. The conclusion from all these experiment is that FXYD1 raises the binding affinity of  α1β1 for Na ions, presumably at the third Na-selective binding site. In whole cell expression studies FXYD1 reduces the apparent affinity  for Na ions. Possible reasons for the difference from this study using the purified recombinant Na,K-ATPase are discussed.</dcterms:abstract>
    <dc:contributor>Mishra, Neeraj Kumar</dc:contributor>
    <dc:contributor>Cirri, Erica</dc:contributor>
    <dcterms:bibliographicCitation>First publ. in: Biochemistry ; 50 (2011), 18. - S. 3736-3748</dcterms:bibliographicCitation>
    <dc:creator>Katz, Adriana</dc:creator>
    <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/>
    <dc:contributor>Belogus, Talya</dc:contributor>
    <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2012-05-31T22:25:05Z</dcterms:available>
    <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/>
    <dc:creator>Lifshitz, Yael</dc:creator>
    <foaf:homepage rdf:resource="http://localhost:8080/"/>
    <dc:creator>Cirri, Erica</dc:creator>
    <dc:creator>Belogus, Talya</dc:creator>
    <dc:contributor>Karlish, Steven J. D.</dc:contributor>
    <dcterms:rights rdf:resource="https://rightsstatements.org/page/InC/1.0/"/>
    <dc:creator>Karlish, Steven J. D.</dc:creator>
    <dc:language>eng</dc:language>
    <dc:rights>terms-of-use</dc:rights>
    <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/14178"/>
    <dc:contributor>Lifshitz, Yael</dc:contributor>
    <dspace:hasBitstream rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/14178/2/apell_phospholemman.pdf"/>
    <dc:contributor>Garty, Haim</dc:contributor>
    <dcterms:issued>2011-05-10</dcterms:issued>
    <dc:creator>Garty, Haim</dc:creator>
    <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/28"/>
    <dcterms:hasPart rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/14178/2/apell_phospholemman.pdf"/>
    <dc:contributor>Katz, Adriana</dc:contributor>
  </rdf:Description>
</rdf:RDF>
Interner Vermerk
xmlui.Submission.submit.DescribeStep.inputForms.label.kops_note_fromSubmitter
Kontakt
URL der Originalveröffentl.
Prüfdatum der URL
Prüfungsdatum der Dissertation
Finanzierungsart
Kommentar zur Publikation
Allianzlizenz
Corresponding Authors der Uni Konstanz vorhanden
Internationale Co-Autor:innen
Universitätsbibliographie
Ja
Begutachtet
Diese Publikation teilen