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Epitope topology of Na,K‐ATPase α subunit analyzed in basolateral cell membranes of rat kidney tubules
Author(s) -
Fujimoto Kazushi,
Møller Jesper Vuust,
Maunsbach Arvid B.
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)01002-2
Subject(s) - immunogold labelling , epitope , membrane , immunoelectron microscopy , cytoplasm , microbiology and biotechnology , immunocytochemistry , chemistry , extracellular , cell membrane , biology , biophysics , biochemistry , antibody , ultrastructure , anatomy , immunology , endocrinology
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 α 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 (Gly 1 ‐His 13 ), Leu 815 ‐Gln 828 and the C‐terminus (Ile 1002 ‐Tyr 1006 ) 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 Asn 889 ‐Gln 903 to cross‐fractured basolateral plasma membranes suggesting that this epitope is located on the extracellular side of the membrane.