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 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.
Vladimirova
N M
NM
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow.
Potapenko
N A
NA
Sachs
G
G
Modyanov
N N
NN
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000648:ultrastructure; D002478:Cells, Cultured; D066298:In Vitro Techniques; D007668:Kidney; D007784:Lactoperoxidase / Q000737:chemistry; D008565:Membrane Proteins / Q000648:ultrastructure; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D000254:Sodium-Potassium-Exchanging ATPase / Q000648:ultrastructure*; D013552:Swine; D014443:Tyrosine / Q000737:chemistry
D008565:Membrane Proteins; D014443:Tyrosine; D007784:Lactoperoxidase; D000254:Sodium-Potassium-Exchanging ATPase