Epitope topology of Na,K-ATPase alpha subunit analyzed in basolateral cell membranes of rat kidney tubules.
FEBS Lett
; 395(1): 29-32, 1996 Oct 14.
Article
en En
| MEDLINE
| ID: mdl-8849683
ABSTRACT
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.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Membrana Celular
/
ATPasa Intercambiadora de Sodio-Potasio
/
Túbulos Renales
/
Epítopos
Límite:
Animals
Idioma:
En
Revista:
FEBS Lett
Año:
1996
Tipo del documento:
Article
País de afiliación:
Japón