Topology of Na+,K+-ATPase. Identification of the extra- and intracellular hydrophilic loops of the catalytic subunit by specific antibodies.
To study the topology of Na+,K+-ATPase monoclonal antibodies (MAbs) specific for membrane-bound enzyme were produced. Using immunofluorescence staining of viable cells or smears of a pig kidney embryonic (PKE) cell line, two groups of MAbs were selected, namely those binding to extra- or intracellular portions of the alpha-subunit. The extracellular location of peptide loop 804-841 linking the Vth and VIth intramembrane hydrophobic segments was proved using MAb VG2. Another MAb, IIC9, interacting with PKE cells only after membrane perforation (4% formaldehyde and 0.1% Tween-20), was shown to bind to the hydrophilic loop 868-945. The antigenic determinants recognized by MAb IIC9 and VG2 are located in peptides 887-904 and 810-825, respectively. The C-terminus of the alpha-subunit molecule was positioned on the outer side of the cytoplasmic membrane utilizing affinity-purified antibodies to the synthetic peptide corresponding to fragment 999-1008.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002462:Cell Membrane / Q000201:enzymology; D000939:Epitopes / Q000032:analysis; D007679:Kidney Medulla / Q000201:enzymology; D046911:Macromolecular Substances; D008958:Models, Molecular; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000276:immunology; D013552:Swine
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D000254:Sodium-Potassium-Exchanging ATPase