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Topology of Na,K‐ATPase α subunit epitopes analyzed with oligopeptide‐specific antibodies and double‐labeling immunoelectron microscopy
Author(s) -
Ning Gang,
Maunsbach Arvid B.,
Lee Yang-Jae,
Møller Jesper Vuust
Publication year - 1993
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(93)80868-u
Subject(s) - epitope , immunoelectron microscopy , oligopeptide , polyclonal antibodies , microbiology and biotechnology , protein subunit , transmembrane protein , biology , antibody , cytoplasm , chemistry , extracellular , biochemistry , peptide , receptor , immunology , gene
Using four oligopeptide‐specific polyclonal antibodies, we mapped the α subunit ofNa,K‐ATPase by double‐labeling immunoelectron microscopy combined with negative staining. The results show that the epitopes of the N‐tenninus (Gly 1 ‐His 13 ), C‐terminus (Ile 1002 ‐Tyr 1016 ) and Leu 815 ‐Gln 828 are located on the same face of crystallized Na,K‐ATPase membranes from pig kidney, whereas the epitope Asn 889 ‐Gln 903 is present on the opposite side. The present study demonstrates the cytoplasmic location of C‐terminus and that Leu 828 ‐Gln 828 is exposed on the eytoplasmic and Asn 889 ‐Gln 903 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 Leu 815 ‐Gln 828 and Asn 889 ‐Gln 903 .