Na,K-ATPase extracellular surface probed with a monoclonal antibody that enhances ouabain binding.
The Na,K-stimulated ATPase is inhibited by extracellular cardiac glycosides, which bind to the enzyme's alpha subunit. We used a monoclonal antibody, VG4, as a probe of the extracellular surface. The antibody was specific for Na,K-ATPase and bound to intact cells. The epitope was mapped to the first extracellular loop (H1-H2) of alpha, using a combination of techniques including trypsinolysis, N-terminal sequence of a fragment containing the determinant, and analysis of the effects of species-specific sequence differences. The antibody inhibited Na,K-ATPase activity under certain circumstances, indicating that the H1-H2 loop participates in conformational changes that are transmitted to the active site. Mutations in the H1-H2 loop have been shown by others to affect ouabain affinity. Ouabain and the antibody acted synergistically to inhibit the enzyme, which seemingly supported the hypothesis that the H1-H2 loop is an essential part of the cardiac glycoside binding site. Direct measurements of the binding of [3H]ouabain, however, indicated that VG4 enhanced rather than inhibited binding, presumably by promoting favorable conformation changes. The data suggest the possibility that the cardiac glycoside binding site may be intramembrane rather than extracellular.
Arystarkhova
E
E
Department of Neurosurgical Research, Massachusetts General Hospital, Boston 02114.
Gasparian
M
M
Modyanov
N N
NN
Sweadner
K J
KJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001666:Binding Sites, Antibody; D002301:Cardiac Glycosides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D010042:Ouabain / Q000378:metabolism*; D011487:Protein Conformation; D016415:Sequence Alignment; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D013552:Swine
D000911:Antibodies, Monoclonal; D002301:Cardiac Glycosides; D000939:Epitopes; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase