Published Online
on July 15, 2002

A more recent version of this article appeared on August 1, 2002

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Submitted on May 13, 2002
Revised on June 3, 2002

Ouabain-Binding Protein(s) From Human Plasma

Behnaz Parhami-Seren; Richard Haberly; Michael N. Margolies; and Garner T. Haupert Jr^*

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.

^* To whom correspondence should be addressed. E-mail: haupert.garner{at}mgh.harvard.edu.

Abstract—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 (IC₅₀4 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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