A SPECIFIC BINDING-PROTEIN FOR CARDIAC-GLYCOSIDES EXISTS IN BOVINE SERUM

Searching for a binding protein in blood, which may be involved in the specific transport of cardiac glycosides to their receptor sites on t he sodium pump, we isolated a cardiac glycoside-binding protein (CGBG) of 26 kDa from the globulin fraction of bovine serum by affinity chro matography and on a ouabain-Sepharose 4B column by a purification fact or of 5000. The cardiac glycoside-binding globulin was labeled specifi cally and covalently by the protein-reactive digoxigenin derivative HD MA xigenin-3-O-methylcarbonyl-epsilon-aminocaproate). Even very high c oncentrations of other steroids, such as estrogen, testosterone, proge sterone, and cortisone, did not prevent HDMA-labeling (at 5 and 100 nM ) of CGBG, but the cardenolides ouabain and digoxin or the bufadienoli de proscillaridin A did so. CGBG is a homodimer of two 26-kDa subunits forming disulfide bonds, since HDMA labeling of a protein of 53 kDa w as observed in SDS-polyacrylamide gel electrophoresis when beta-mercap toethanol was absent during SDS denaturation, The N-terminal amino aci d sequence K-D-V-Y-R-A-P-D-G-T-Q-S-A showed no sequence similarity wit h proteins recorded in gene and protein sequence data banks. A 90-kDa cytosolic CGBG exists in bovine kidneys and reacts with antibodies aga inst CGBG. Binding of ouabain to the cardiac glycoside-binding globuli n was monitored by quenching of intrinsic tryptophan fluorescence. Suc h studies reveal two negatively cooperative ouabain binding sites with K-d' of 1.52 nM and K-d'' = 75 nM and with an interaction factor of 5 0 using a Koshland-Nemethy-Filmer model. The demonstration of a cardia c glycoside-binding globulin in plasma is consistent with the recent f inding of endogenous cardiac glycosides in mammals.