Topology of Na,K‐ATPase α subunit epitopes analyzed with oligopeptide‐specific antibodies and double‐labeling immunoelectron microscopy

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Abstract
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 (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 Leu828‐Gln828 is exposed on the eytoplasmic 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.