Ouabain-Binding Protein(s) From Human Plasma

Abstract

Abstract-Conservation of the binding site on mammalian Na ϩ ,K ϩ -ATPase for cardiac glycosides and the importance of the Na ϩ 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 IC 50 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 C 18 inhibit Na ϩ pump activity in human erythrocytes (IC 50Ϸ 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 (Յ0.5% of total protein A immunoglobulin) that function as a reservoir and delivery system for ouabain-like compounds in the modulation of human Na ϩ ,K ϩ -ATPase in vivo. (Hypertension. 2002;40:220-228.)

Ouabain-Like Compound Changes Rapidly on Physical Exercise in Humans and Dogs

“…No apparent toxicity was observed, however, in the present study. Hence, either the actual free OLC concentration in plasma is lower than that one determined after extraction (eventually caused by its binding to cardiac glycoside binding proteins in blood 20,21 ) or the sodium pump is not yet inhibited within the time interval studied because of the slow on-rate in forming the inhibitory and almost irreversible ouabain-Na ϩ /K ϩ -ATPase complex. 22 A transient increase of endogenous ouabain may not lead to inhibition of the pump, although a rapidly dissociable ouabain-Na ϩ /K ϩ -ATPase complex might be formed.…”

Ouabain as a Mammalian Hormone

“…Serum albumin is considered so far to fulfill the transport function for cardiotonic steroids in blood,18 but the dissociation constants of the cardiac glycoside albumin complexes are well above therapeutic digoxin concentrations. Recent search for a specific cardiac glycoside binding protein in serum resulted in the isolation of several proteins with high affinity for ouabain, that is, a protein of 50 kDa with unknown amino acid sequence and function;17 a 14.4‐kDa protein representing the plasmin‐cleaved carboxy‐terminal end of the heavy chain of IgG2 and IgG1;19 and an immunoglobulin‐like protein with bands of 80, 50, and 25 kDa in SDS‐PAGE representing ≤ 0.5% of the IgG fraction 20. In our trials to characterize the previously described cardiac glycoside binding protein from bovine serum17 any further, we were confronted with the observation that, in addition to the formerly described 50‐kDa band, a 90‐kDa protein also became visible in affinity labeling with HDMA.…”

Endogenous Cardiotonic Steroids: Physiology, Pharmacology, and Novel Therapeutic Targets

“…Subsequently, Antolovic et al (1998) isolated from mammalian plasma and kidneys a specific cardiac glycoside-binding globulin with higher- and lower-affinity binding sites for ouabain and possibly for endogenous CTS. Later, CTS-binding protein was identified as a fragment of IgG (Komiyama et al, 1998; Parhami-Seren et al, 2002). The importance of CTS-binding protein(s) on the function of CTS has not been studied consistently yet; however, Antolovic et al (2000) suggested that binding of CTS to and unbinding of CTS from this transport protein may be involved in a rapid and transient 36-fold rise of EO in response to exercise.…”