APPLICATION OF LACTOPEROXIDASE IODINATION METHOD FOR TOPOGRAPHICAL STUDY OF THE NA-ATPASE CATALYTIC SUBUNIT(, K+)

The orientation of the carboxy terminal pair of tyrosine residues of t he Na+, K+-ATPase alpha-subunit in the plane of the plasma membrane wa s determined. The method was based on lactoperoxidase-catalyzed radioi odination of tyrosine residues accessible on the surface of the enzyme molecule in intact cells of a pig kidney embryonic cell line, in plas ma membrane fraction, and in isolated membrane-bound Na+, K+-ATPase. T he labeled alpha-subunit was isolated by SDS gel electrophoresis with the following electroblotting, its identification was performed by imm unostaining with monoclonal antibodies alpha-p999 and N-terminal seque ncing. The C-terminal amino acids were hydrolyzed step-by-step by carb oxypeptidases B and Y. Radioactivity and quantitative analysis of the protein and released amino acids showed that the C-terminal tyrosine r esidues of the alpha-subunit were accessible to modification only when lactoperoxidase had access to the inner side of the plasma membrane. Therefore, the C-terminus of the Na+, K+-ATPase alpha-subunit is locat ed on the cytoplasmic surface of the pump molecule, and its polypeptid e chain must have an even number of transmembrane segments.