Immunoelectron microscopy of epitopes on Na,K-ATPase catalytic subunit. Implications for the transmembrane organization of the C-terminal domain.
The transmembrane folding of the alpha subunit of Na,K-ATPase was studied by using immunoelectron microscopy to determine whether monoclonal antibodies with defined epitopes bind to the extracellular or cytoplasmic surface. In double labeling experiments, an antibody and a reference marker were bound to purified membrane-associated Na,K-ATPase and were visualized by employing colloidal gold particles of two different sizes. Wheat germ agglutinin and a previously characterized monoclonal antibody were used as control markers for the exoplasmic and cytoplasmic surfaces, respectively. Three antibodies, VG4, VG2, and IIC9, unambiguously bound to the extracellular surface. Previously IIC9 had been assigned to the cytoplasmic surface because, in immunofluorescence studies, it stained intact cells only when they were detergent-permeabilized. To investigate the basis for this contradiction, a third assay for sidedness was used: competition binding in solution to right-side-out renal medullary vesicles. IIC9 was found to bind to the extracellular surface of sealed vesicles, but only in certain experimental conditions. It was concluded that IIC9 has an epitope that is not always accessible and that in this instance, studies of binding to intact and detergent-treated cells had given misleading results. An extracellular disposition for all three antibodies is not compatible with existing folding models, and new models are presented.
Mohraz
M
M
Department of Cell Biology, New York University School of Medicine, New York 10016.
Arystarkhova
E
E
Sweadner
K J
KJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001666:Binding Sites, Antibody; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D007425:Intracellular Membranes / Q000201:enzymology* / Q000648:ultrastructure; D007679:Kidney Medulla / Q000201:enzymology / Q000648:ultrastructure; D007700:Kinetics; D046911:Macromolecular Substances; D016253:Microscopy, Immunoelectron / Q000379:methods; D008861:Microsomes / Q000201:enzymology / Q000648:ultrastructure; D008969:Molecular Sequence Data; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D013552:Swine
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D000254:Sodium-Potassium-Exchanging ATPase