Immunochemical evidence that the FITC-labeling site on Na+,K+-ATPase is not the ATP binding site.
The covalent labeling of the alpha subunit of lamb kidney Na+,K+-ATPase by fluorescein 5'-isothiocyanate at Lys-501 has generally been assumed to occur at the ATP binding site. We have found that the peptide sequence 496HLLVMKGAPER506 serves as the antigenic determinant for monoclonal antibody M8-P1-A3. This antibody binds to both native and FITC-labeled enzyme and while this epitope undergoes ligand-induced changes these changes are not involved in either enzyme function or the E1 in equilibrium E2 conformational changes monitored by FITC-fluorescence intensity.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001667:Binding, Competitive; D000939:Epitopes / Q000032:analysis; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000276:immunology / Q000378:metabolism*; D013861:Thiocyanates / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005452:Fluoresceins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D013861:Thiocyanates; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate