Cytoplasmic segment interactions in the alpha1-subunit of the rat Na+, K+-atpase.
The currently accepted topographical model for the organization of the alpha-subunit of the Na+, K+-ATPase in the membrane considers that the protein has ten transmembrane segments and six cytoplasmic loops. Evidence of interaction between the cytoplasmic regions may contribute to a better understanding of the structure/function relationship of this protein. In this study, the first four cytoplasmic segments (C1, C2, C3 and C4) of the rat alpha1 subunit were expressed in Escherichia Coli. The large cytoplasmic loop between transmembrane segments four and five (C3) retained its native structure as demonstrated by the ability of ATP to protect against chemical modification by Fluorescein 5-isothiocyanate (FITC). Interaction studies were conducted by an overlay assay (Far Western blots) and surface plasmon resonance technology. We observed that C3 interacts with the N-terminal segment of the Na+, K+-ATPase, C1; and that both C1 and C3 interact with the cytoplasmic segments C2 and C4.
Salgado-Commissariat
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Dept of Pharmacological and Pharmaceutical Sciences, University of Houston, TX 77204-5515 USA.
Zandomeni
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Pedemonte
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D000818:Animals; D003593:Cytoplasm / Q000201:enzymology* / Q000502:physiology / Q000528:radiation effects; D018076:DNA, Complementary / Q000502:physiology; D004347:Drug Interactions; D004926:Escherichia coli / Q000502:physiology; D005362:Ficoll / Q000008:administration & dosage / Q000031:analogs & derivatives*; D016650:Fluorescein-5-isothiocyanate / Q000008:administration & dosage / Q000031:analogs & derivatives*; D023421:Models, Animal; D016133:Polymerase Chain Reaction; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D021122:Protein Subunits; D051381:Rats; D017422:Sequence Analysis, DNA; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects / Q000502:physiology*; D020349:Surface Plasmon Resonance
D018076:DNA, Complementary; D021122:Protein Subunits; C045540:fluorescein 5-isothiocyanate-Ficoll; D005362:Ficoll; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate