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Determination of the sidedness of the carboxy-terminus of the Na+/K(+)ATPase alpha-subunit using lactoperoxidase iodination.

Authors
  • Vladimirova, N M
  • Potapenko, N A
  • Sachs, G
  • Modyanov, N N
Type
Published Article
Journal
Biochimica et Biophysica Acta
Publisher
Elsevier
Publication Date
Feb 15, 1995
Volume
1233
Issue
2
Pages
175–184
Identifiers
PMID: 7865541
Source
Medline
License
Unknown

Abstract

The orientation of the carboxy-terminal pair of tyrosines of the Na+/K(+)-ATPase alpha-subunit with respect to the plane of the plasma membrane was determined. The approach was based on lactoperoxidase-catalysed radioiodination of the tyrosine residues accessible on the surface of the enzyme molecule in intact cells of a pig kidney embryonic cell line and those accessible in a broken plasma membrane fraction and in isolated membrane-bound Na+/K(+)-ATPase. The labeled alpha-subunit was isolated by SDS gel electrophoresis followed by electroblotting. Then the COOH-terminal amino acids were hydrolyzed by carboxypeptidases B and Y. Radioactivity and quantitative analysis of the protein and released amino acids showed that the COOH-terminal tyrosine residues of the alpha-subunit were only accessible to modification only when lactoperoxidase had access to the inner side of the plasma membrane. Therefore, the COOH-terminus of the Na+/K(+)-ATPase alpha-subunit is located on the cytoplasmic surface of the pump molecule and its polypeptide chain must have an even number of transmembrane segments.

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