Identification of a Model Cardiac Glycoside Receptor:  Comparisons with Na+,K+-ATPase

• Published in: Biochemistry (Easton) Vol. 37; no. 19; pp. 6658 - 6666
• Main Authors: Kasturi, Rama, Yuan, Jie, McLean, Larry R, Margolies, Michael N, Ball, William J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 12-05-1998
• Subjects: Animals; Anthracenes - metabolism; Antibodies, Monoclonal - metabolism; Antibody Affinity; Antigens - metabolism; Cardiac Glycosides - metabolism; Energy Transfer; Fluorescence Polarization; Fluorescent Dyes - metabolism; Immunoglobulin Fab Fragments - metabolism; Kidney Medulla - enzymology; Kinetics; Models, Molecular; Ouabain - analogs & derivatives; Ouabain - metabolism; Protein Binding; Sheep; Sodium-Potassium-Exchanging ATPase - chemistry; Sodium-Potassium-Exchanging ATPase - metabolism; Spectrometry, Fluorescence
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi973037d

The availability of high-affinity anti-digoxin monoclonal antibodies (mAbs) offers the potential for their use as models for the characterization of the relationship between receptor structure and cardiac glycoside binding. We have characterized the binding of anthroylouabain (AO), a fluorescent derivative of the cardiac glycoside ouabain, to mAbs 26-10, 45-20, and 40-50 [Mudgett-Hunter, M., et al. (1995) Mol. Immunol. 22, 477] and lamb kidney Na+,K+-ATPase by monitoring the resultant AO fluorescence emission spectra, anisotropy, lifetime values, and Förster resonance energy transfer (FRET) from protein tryptophan(s) (Trp) to AO. These data suggest that the structural environment in the vicinity of the AO-binding site of Na+,K+-ATPase is similar to that of mAb 26-10 but not mAbs 45-20 and 40-50. A model of AO complexed to the antigen binding fragment (Fab) of mAb 26-10 which was generated using known X-ray crystal structural data [Jeffrey, P. D., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10310] shows a heavy chain Trp residue (Trp-H100) that is close (∼3 Å) to the anthroyl moiety. This is consistent with the energy transfer seen upon AO binding to mAb 26-10 and suggests that Trp-H100, which is part of the antibody's cardiac glycoside binding site, is a major determinant of the fluorescence properties of bound AO. In contrast, the generated model of AO complexed to Fab 40-50 [Jeffrey, P. D., et al. (1995) J. Mol. Biol. 248, 344] shows a heavy chain Tyr residue (Tyr-H100) which is part of the cardiac glycoside binding site, located ∼10 Å from the anthroyl moiety. The closest Trp residues (H52 and L35) are located ∼17 Å from the anthroyl moiety, and no FRET is observed despite the fact that these Trp residues are close enough for significant FRET to occur. The energy transfer seen upon AO binding to Na+,K+-ATPase suggests the presence of one completely quenched or two highly quenched enzyme Trp residues ∼10 and ∼17 Å, respectively, from the anthroyl moiety. These data suggest that the Na+,K+-ATPase Trp residue(s) involved in fluorescence energy transfer to AO is likely to be part of the cardiac glycoside binding site.