Determination of the sidedness of the carboxy-terminus of the Na+/K+-ATPase α-subunit using lactoperoxidase iodination
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES(1995)
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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Key words
ATPASE, NA+/K+-,MEMBRANE TOPOLOGY,IODINATION
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