FEBS Letters | |
Epitope topology of Na,K‐ATPase α subunit analyzed in basolateral cell membranes of rat kidney tubules | |
Maunsbach, Arvid B.3  Fujimoto, Kazushi1  Møller, Jesper Vuust2  | |
[1] Department of Anatomy, Faculty of Medicine, Kyoto University, Yoshida-Konoecho, Sakyo-ku, Kyoto 606-01, Japan;Institute of Biophysics at the Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark;Department of Cell Biology at the Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark | |
关键词: Na; K-ATPase; Membrane epitope; Topology; Freeze-fracture replica labeling; Immunoelectron microscopy; Oligopeptide-specific antibody; | |
DOI : 10.1016/0014-5793(96)01002-2 | |
学科分类:生物化学/生物物理 | |
来源: John Wiley & Sons Ltd. | |
【 摘 要 】
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 (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.
【 授权许可】
Unknown
【 预 览 】
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RO201912020303349ZK.pdf | 665KB | download |