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