ubiquinone and 5--adenylyl-(beta-gamma-methylene)diphosphonate

FXYD domain-containing proteins are tissue-specific regulators of the Na,K-ATPase that have been shown to have significant physiological implications. Information about the sites of interaction between some FXYD proteins and subunits of the Na,K-ATPase is beginning to emerge. We previously identified an FXYD protein in plasma membranes from shark rectal gland cells and demonstrated that this protein (FXYD10) modulates shark Na,K-ATPase activity. The present study was undertaken to identify the location of the C-terminal domain of FXYD10 on the alpha-subunit of Na,K-ATPase, using covalent cross-linking combined with proteolytic cleavage. Treatment of Na,K-ATPase-enriched membranes with the homobifunctional thiol cross-linker 1,4-bismaleimidyl-2,3-dihydroxybutane resulted in cross-linking of FXYD10 to the alpha-subunit. Cross-linking was not affected by preincubation with sodium or potassium but was significantly reduced after pre-incubation with the non-hydrolyzable ATP analog beta,gamma-methyleneadenosine 5'-triphosphate (AMP-PCP). A peptic assay was developed, in which pepsin treatment of Na,K-ATPase at low pH resulted in extensive cleavage of the alpha-subunit while FXYD10 was left intact. Proteolytic fragments of control and cross-linked preparations were isolated by immunoprecipitation and analyzed by gel electrophoresis. A proteolytic fragment containing FXYD10 cross-linked to a fragment from the alpha-subunit could be localized on SDS gels. Sequencing of this fragment showed the presence of FXYD10 as well as a fragment within the A domain of the alpha-subunit comprising 33 amino acids, including a single Cys residue, Cys254. Thus, regulation of Na,K-ATPase by FXYD10 occurs in part via cytoplasmic interaction of FXYD10 with the A domain of the shark alpha-subunit.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Binding Sites; Butylene Glycols; Cell Membrane; Chloride Channels; Cross-Linking Reagents; Cysteine; Cytoplasm; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Fish Proteins; Hydrogen-Ion Concentration; Immunoblotting; Immunoprecipitation; Ligands; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Pepsin A; Phosphoproteins; Phosphorylation; Potassium; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Sodium; Sodium-Potassium-Exchanging ATPase; Squalus acanthias; Sulfhydryl Compounds; Ubiquinone