Topology of Na,K-ATPase alpha subunit epitopes analyzed with oligopeptide-specific antibodies and double-labeling immunoelectron microscopy.
Using four oligopeptide-specific polyclonal antibodies, we mapped the alpha subunit of Na,K-ATPase by double-labeling immunoelectron microscopy combined with negative staining. The results show that the epitopes of the N-terminus (Gly1-His13), C-terminus (Ile1002-Tyr1016) and Leu815-Gln828 are located on the same face of crystallized Na,K-ATPase membranes from pig kidney, whereas the epitope Asn889-Gln903 is present on the opposite side. The present study demonstrates the cytoplasmic location of C-terminus and that Leu815-Gln828 is exposed on the cytoplasmic and Asn889-Gln903 on the extracellular side. The results are consistent with an eight- or ten-segment model, and support the existence of an M5/M6 loop and the presence of one transmembrane segment between Leu815-Gln828 and Asn889-Gln903.
Ning
G
G
Department of Cell Biology, Institute of Anatomy, University of Aarhus, Denmark.
Maunsbach
A B
AB
Lee
Y J
YJ
Møller
J V
JV
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D007106:Immune Sera; D007679:Kidney Medulla / Q000201:enzymology; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000276:immunology; D011817:Rabbits / Q000276:immunology; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis* / Q000276:immunology; D013552:Swine
D000906:Antibodies; D000939:Epitopes; D007106:Immune Sera; D009842:Oligopeptides; D000254:Sodium-Potassium-Exchanging ATPase