title: Phospholemman (FXYD1) raises the affinity of the human α1β1 isoform of Na,K-ATPase for Na ions.
abstract: The human α(1)/His(10)-β(1) 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 α(1)β(1) 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 α(1)/His(10)-β(1) complex and stabilizes it in an active mode. The functional properties of the α(1)/His(10)-β(1) and α(1)/His(10)-β(1)/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 E(1) and P-E(2) 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, Na(3)E(1)-P → P-E(2)Na(3). No significant differences with or without FXYD1 could be detected. Rate constants of the conformational transitions Rb(2)E(1) → E(2)(Rb(2)) and E(2)(Rb(2)) → Na(3)E(1), investigated with fluorescein-labeled Na,K-ATPase, showed only minor or no effects of FXYD1, respectively. The conclusion from all these experiments is that FXYD1 raises the binding affinity of α(1)β(1) 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.
authors: Department of Biology, University of Konstanz, 78464 Konstanz, Germany.
authors: Erica
authors: E
authors: Cirri
authors: Adriana
authors: A
authors: Katz
authors: Neeraj Kumar
authors: NK
authors: Mishra
authors: Talya
authors: T
authors: Belogus
authors: Yael
authors: Y
authors: Lifshitz
authors: Haim
authors: H
authors: Garty
authors: Steven J D
authors: SJ
authors: Karlish
authors: Hans-Jürgen
authors: HJ
authors: Apell
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007477:Ions; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D010750:Phosphoproteins / Q000737:chemistry / Q000502:physiology*; D010843:Pichia / Q000378:metabolism; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry; D012964:Sodium / Q000737:chemistry*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: C503551:ATP1B1 protein, human; D007477:Ions; D008565:Membrane Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D012709:Serum Albumin; C069141:phospholemman; D000255:Adenosine Triphosphate; D012964:Sodium; C499076:ATP1A1 protein, human; D000254:Sodium-Potassium-Exchanging ATPase