Evidence for the organization of the transmembrane segments of (Na,K)-ATPase based on labeling lipid-embedded and surface domains of the alpha-subunit.
The purpose of this work has been to examine the organization of the intramembranous portion of the alpha-subunit of membrane-bound (Na,K)-ATPase. Covalent labeling of the alpha-subunit and its tryptic fragments from within the lipid bilayer with [125I]iodonaphthylazide was combined with covalent labeling with 32P from [gamma-33P]ATP at the cytoplasmic surface and with [3H]N-(ouabain)-N'-(2-nitro-4-azidophenyl)ethylenediamine from the extra cellular surface. In control experiments using extensive proteolysis and reduced glutathione, it is confirmed that iodonaphthylazide labels segments of the protein within the lipid bilayer. The labeled segments of the alpha-subunit, produced by extensive proteolysis, are selectively extracted by organic solvents. Both at a low and at a high concentration of iodonaphthylazide, about 50% of label added to the medium is covalently attached to protein and lipid. At the low iodonaphthylazide concentration, the NH2-terminal Mr = 46,000 (46K) fragment of the alpha-subunit is preferentially labeled, while at the higher concentration of the 46K fragment, the 78K fragment, and the COOH-terminal 58K fragment are labeled. 32P from [gamma-32P]ATP is incorporated into the 46K fragment while [3H]N-(ouabain)-N'-(2-nitro-4-azido-phenyl)ethylenediamine from the extracellular surface labels all the major fragments, 78K, 58K, and 46K. The data provide evidence for a model of the path of the polypeptide chain with multiple traverses of the alpha-subunit across the bilayer and the NH2-terminal and three trypsin-sensitive bonds exposed at the cytoplasma surface.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D005978:Glutathione / Q000494:pharmacology; D007679:Kidney Medulla / Q000201:enzymology*; D008051:Lipid Bilayers; D046911:Macromolecular Substances; D008563:Membrane Lipids / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine
D008051:Lipid Bilayers; D046911:Macromolecular Substances; D008563:Membrane Lipids; D010446:Peptide Fragments; D000254:Sodium-Potassium-Exchanging ATPase; D005978:Glutathione