Ouabain-binding protein(s) from human plasma.
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 approximately 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 (< or =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.
Parhami-Seren
Behnaz
B
Department of Surgery, Medical Services, Massachusetts General Hospital, Harvard Medical School, Boston, Mass 02129, USA.
Haberly
Richard
R
Margolies
Michael N
MN
Haupert
Garner T
GT
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins / Q000097:blood* / Q000302:isolation & purification / Q000494:pharmacology; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains; D010042:Ouabain / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D012414:Rubidium Radioisotopes / Q000493:pharmacokinetics; D012685:Sepharose / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism; D005719:gamma-Globulins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D012414:Rubidium Radioisotopes; D005719:gamma-Globulins; D010042:Ouabain; D012685:Sepharose; D000254:Sodium-Potassium-Exchanging ATPase