Immunochemical identification of exposed regions of the Na+,K(+)-ATPase alpha-subunit.
A polyclonal antibody against the Na+,K(+)-ATPase holoenzyme was prepared. This antibody recognized native Na+,K(+)-ATPase and inhibited its activity. The peptide fragments corresponding to various regions of the Na+,K(+)-ATPase alpha-subunit, which were synthesized from the cDNA, were immunoprecipitated with the antibody, and the M32-D75, M158-D197 and M470-V552 fragments (the latter included K508, a putative ATP binding site) were identified as the epitopes. The M267-I442 fragment, which included a phosphorylation site at D376, and the C-terminal one-third of the alpha-subunit from M615 to the C-terminus, were not detected using this antibody. These results suggest that at least three regions of the Na+,K(+)-ATPase alpha-subunit, M32-D75, M158-D197 and M470-V552, cover its exposed regions, and that some of them are essential for ATPase activity.
Homareda
H
H
Department of Biochemistry, Kyorin University School of Medicine, Tokyo, Japan.
Nagano
Y
Y
Matsui
H
H
D000818:Animals; D001666:Binding Sites, Antibody; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D007120:Immunochemistry / Q000379:methods; D007074:Immunoglobulin G / Q000276:immunology; D007668:Kidney / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010766:Phosphorylation; D010957:Plasmids; D011233:Precipitin Tests; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection
D000939:Epitopes; D007074:Immunoglobulin G; D010446:Peptide Fragments; D000254:Sodium-Potassium-Exchanging ATPase