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Abstract

Mammals, including man, appear to have in the circulation a compound with biological properties similar to those of the plant-derived cardiotonic steroids like digitalis or ouabain. Endogenous ouabain (Oua) or “ouabain-like compound” (OLC) binds to the Na+, K+-ATPase (Na+ pump) with high affinity and has been implicated in endogenous regulation of Na+ and K+ transport as well as the pathogenesis of human essential hypertension. The form in which OLC circulates is unknown and the existence of a carrier protein has been raised. We produced monoclonal antibodies (mAbs) to Oua which recognize Oua with high affinity (10-8 M), but not digoxin (Dig), the glycoside in prevalent clinical use. Sepharose-bound mAb was used as an affinity column to screen human plasma. Column eluates were found to contain OLC and an associated protein (major band ~ 80 kD). The protein shows specificity because it binds Oua with high affinity (Ka~200 nM), but not Dig. Fractions containing OLC binding protein (OLC-BP) are physiologically relevant because they inhibited active Na+ transport in human tissues, confirming the presence of OLC. OLC-BP reacted with anti-human immunoglobulin (Ig) but not anti-mouse Ig, confirming human origin and suggesting OLC-BP to be immunoglobulin-like. Yield of OLC-BP could be enhanced by purifying human plasma first with protein A (binds human Ig) followed by affinity purification with Oua mAb-Sepharose. Immobilized OLC-BP purified thus binds 125I Oua and this binding is inhibited by nanomolar concentrations of free Oua but not Dig.

In summary, an OLC-BP was purified from human plasma. OLC-BP appears to be a protein A binding Ig or Ig-like molecule. It binds Oua specifically with affinity ~200 nM. Fractions containing OLC-BP also contain Na+ pump inhibitory activity presumed to be OLC. It is suggested that our anti-Oua mAb purifies OLC complexed to the binding protein from human plasma. Given the relative affinities of Oua for OLC-BP and cellular Na+, K+-ATPase in vivo, it is possible that OLC-BP could serve as a reservoir and delivery system, releasing OLC to the Na+ pump in target tissues.

ouabain-like compound, sodium-potassium ATPase, ouabain monoclonal antibodies
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© American Journal of Hypertension, Ltd. 2001
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