DETERMINATION OF THE SIDEDNESS OF THE CARBOXY-TERMINUS OF THE NA+ K+-ATPASE ALPHA-SUBUNIT USING LACTOPEROXIDASE IODINATION/

The orientation of the carboxy-terminal pair of tyrosines of the Na+/K +-ATPase alpha-subunit with respect to the plane of the plasma membran e 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 lin e and those accessible in a broken plasma membrane fraction and in iso lated membrane-bound Na+/K+-ATPase. The labeled alpha-subunit was isol ated 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 alp ha-subunit were only accessible to modification only when lactoperoxid ase had access to the inner side of the plasma membrane. Therefore, th e COOH-terminus of the Na+/K+-ATPase alpha-subunit is located on the c ytoplasmic surface of the pump molecule and its polypeptide chain must have an even number of transmembrane segments.