Ouabain-Binding Protein(s) From Human Plasma

• Published in: Hypertension (Dallas, Tex. 1979) Tex. 1979), 2002-08, Vol.40 (2), p.220-228
• Main Authors: Parhami-Seren, Behnaz, Haberly, Richard, Margolies, Michael N, Haupert, Garner T
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - metabolism; Biological and medical sciences; Blotting, Western; Carrier Proteins - blood; Carrier Proteins - isolation & purification; Carrier Proteins - pharmacology; Cattle; Cell receptors; Cell structures and functions; Electrophoresis, Polyacrylamide Gel; Erythrocytes - drug effects; Erythrocytes - metabolism; Fundamental and applied biological sciences. Psychology; gamma-Globulins - metabolism; Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors; Humans; Mice; Mice, Inbred Strains; Molecular and cellular biology; Ouabain - immunology; Ouabain - metabolism; Protein Binding; Rubidium Radioisotopes - pharmacokinetics; Sepharose - metabolism; Sodium-Potassium-Exchanging ATPase - antagonists & inhibitors; Sodium-Potassium-Exchanging ATPase - metabolism
• ISSN: 0194-911X; 1524-4563
• DOI: 10.1161/01.HYP.0000027134.14160.1D

Conservation of the binding site on mammalian Na,K-ATPase for cardiac glycosides and the importance of the Na pump in mammalian cellular physiology has stimulated the search for a mammalian analog of these plant compounds. One candidate, isolated from brain and blood, appears to be ouabain itself or a closely related isomer, the ouabain-like compound. Little is known about the circulating form. Because human steroid hormones circulate with carrier proteins, we produced a ouabain-specific monoclonal antibody (mAb 1-10) and used it to probe normal human plasma for ouabain-protein carrier complex. Ouabain-like biological activity was isolated in association with protein bands of 80, 50, and 25 kDa. These proteins appear to be human immunoglobulins or immunoglobulin-like because they are recognized by anti-human immunoglobulin antibodies, but not by anti-mouse immunoglobulin antibodies. The protein-containing fractions inhibit the binding of mAb 1-10 to immobilized ouabain, and with further purification on protein A, the immunoglobulin-like protein binds radioactive ouabain with an IC50 of 200 to 600 nmol/L, but binds digoxin with 100-fold less affinity, suggesting specificity for ouabain or its isomer. Active protein fractions after purification on C18 inhibit Na pump activity in human erythrocytes (IC50≈4 nmol/L, ouabain equivalents), and this chromatography appears to dissociate the ouabain-like compound from the immunoglobulin protein(s). These immunoglobulin-like molecules may represent a subset of immunoglobulins (≤0.5% of total protein A immunoglobulin) that function as a reservoir and delivery system for ouabain-like compounds in the modulation of human Na, K-ATPase in vivo.