Published online before print July 15, 2002, doi: 10.1161/01.HYP.0000027134.14160.1D
(Hypertension. 2002;40:220.)
© 2002 American Heart Association, Inc.
Scientific Contributions |
Ouabain-Binding Protein(s) From Human Plasma
From the Department of Surgery (B.P.-S., M.N.M.) and Renal Unit (R.H., G.T.H.), Medical Services, Massachusetts General Hospital, Harvard Medical School, Boston, Mass.
Correspondence to Dr Garner T. Haupert, Jr, Renal Unit, CNY 8, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129. E-mail haupert.garner{at}mgh.harvard.edu
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.
Key Words: ouabain • digitalis-like factor • immunoglobulins • sodium pump
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