Transmembrane Organization of the Na,K-ATPase Determined by Epitope Addition

Research output: Contribution to journalArticle

55 Citations (Scopus)

Abstract

The Na,K-ATPase is a membrane-associated enzyme that establishes the internal Na+/K+ environment of most animal cells. The catalytic (α) subunit of the Na,K-ATPase contains multiple transmembrane segments, but the number and location of these domains has not been clearly established. We have used epitope addition to determine the transmembrane topology of the α subunit. An immunoreactive peptide was inserted into various regions of the cDNA encoding the rat α1 subunit, and the constructs were expressed in transfected mammalian cells. The intra- or extracellular location of the epitiope tags was determined by immunofluorescence analysis. Our results indicate that the amino and carboxyl termini of the α subunit are situated intracellularly, and the polypeptide is likely to possess eight membrane-spanning segments. The systematic application of epitope tagging may be useful for analyzing the topology of membrane proteins of unknown structure.

Original languageEnglish (US)
Pages (from-to)13782-13786
Number of pages5
JournalBiochemistry
Volume32
Issue number50
DOIs
StatePublished - Jan 1 1993

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Adenosine Triphosphatases
Epitopes
Cells
Topology
Membranes
Peptides
Fluorescent Antibody Technique
Rats
Catalytic Domain
Membrane Proteins
Animals
Complementary DNA
Enzymes
sodium-translocating ATPase

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "Transmembrane Organization of the Na,K-ATPase Determined by Epitope Addition",
abstract = "The Na,K-ATPase is a membrane-associated enzyme that establishes the internal Na+/K+ environment of most animal cells. The catalytic (α) subunit of the Na,K-ATPase contains multiple transmembrane segments, but the number and location of these domains has not been clearly established. We have used epitope addition to determine the transmembrane topology of the α subunit. An immunoreactive peptide was inserted into various regions of the cDNA encoding the rat α1 subunit, and the constructs were expressed in transfected mammalian cells. The intra- or extracellular location of the epitiope tags was determined by immunofluorescence analysis. Our results indicate that the amino and carboxyl termini of the α subunit are situated intracellularly, and the polypeptide is likely to possess eight membrane-spanning segments. The systematic application of epitope tagging may be useful for analyzing the topology of membrane proteins of unknown structure.",
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Transmembrane Organization of the Na,K-ATPase Determined by Epitope Addition. / Canfield, Victor A.; Levenson, Robert.

In: Biochemistry, Vol. 32, No. 50, 01.01.1993, p. 13782-13786.

Research output: Contribution to journalArticle

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AB - The Na,K-ATPase is a membrane-associated enzyme that establishes the internal Na+/K+ environment of most animal cells. The catalytic (α) subunit of the Na,K-ATPase contains multiple transmembrane segments, but the number and location of these domains has not been clearly established. We have used epitope addition to determine the transmembrane topology of the α subunit. An immunoreactive peptide was inserted into various regions of the cDNA encoding the rat α1 subunit, and the constructs were expressed in transfected mammalian cells. The intra- or extracellular location of the epitiope tags was determined by immunofluorescence analysis. Our results indicate that the amino and carboxyl termini of the α subunit are situated intracellularly, and the polypeptide is likely to possess eight membrane-spanning segments. The systematic application of epitope tagging may be useful for analyzing the topology of membrane proteins of unknown structure.

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