Epitope topology of Na,K-ATPase alpha subunit analyzed in basolateral cell membranes of rat kidney tubules.
For topological analysis of integral membrane protein in situ, we used a novel immunoelectron microscopic technique, SDS-digested freeze-fracture replica labeling (SDS-FRL), and oligopeptide-specific antibodies to clarify the sidedness of Na,K-ATPase alpha subunit epitopes in basolateral cell membranes of kidney tubules. Unfixed tissue slices from rat kidney outer medulla were frozen with liquid helium, freeze-fractured, and replicated. After digestion with SDS to solubilize unfractured membranes and cytoplasm, the platinum/carbon replicas, along with attached cytoplasmic and exoplasmic membrane halves, were processed for immunocytochemistry. Immunogold labeling using antibodies against the N-terminus (Gly1-His13), Leu815-Gln828 and the C-terminus (Ile1002-Tyr1006) was superimposed on the images of the electron microscope protoplasmic fracture face of the basolateral plasma membranes, thus demonstrating cytoplasmic locations of these epitopes. On the contrary, SDS-FRL showed specific binding of Asn889-Gln903 to cross-fractured basolateral plasma membranes suggesting that this epitope is located on the extracellular side of the membrane.
Fujimoto
K
K
Department of Anatomy, Faculty of Medicine, Kyoto University, Sakyo-ku, Japan.
Móller
J V
JV
Maunsbach
A B
AB
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology* / Q000648:ultrastructure; D016764:Cell Polarity; D003593:Cytoplasm; D003902:Detergents; D004848:Epithelium / Q000648:ultrastructure; D000939:Epitopes / Q000032:analysis*; D005614:Freeze Fracturing / Q000379:methods; D007684:Kidney Tubules / Q000166:cytology / Q000201:enzymology*; D009842:Oligopeptides; D051381:Rats; D012967:Sodium Dodecyl Sulfate; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*
D003902:Detergents; D000939:Epitopes; D009842:Oligopeptides; D012967:Sodium Dodecyl Sulfate; D000254:Sodium-Potassium-Exchanging ATPase