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 the sodium pump, we isolated a cardiac glycoside-binding protein (CGBG) of 26 kDa from the globulin fraction of bovine serum by affinity chromatography and on a ouabain-Sepharose 4B column by a purification factor of 5000. The cardiac glycoside-binding globulin was labeled specifically and covalently by the protein-reactive digoxigenin derivative HDMA (N-hydroxysuccimidyldigoxigenin-3-O-methylcarbonyl-epsilon-+ ++aminocapro ate). Even very high concentrations of other steroids, such as estrogen, testosterone, progesterone, and cortisone, did not prevent HDMA-labeling (at 5 and 100 nM) of CGBG, but the cardenolides ouabain and digoxin or the bufadienolide 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 was observed in SDS-polyacrylamide gel electrophoresis when beta-mercaptoethanol was absent during SDS denaturation. The N-terminal amino acid sequence K-D-V-Y-R-A-P-D-G-T-Q-S-A showed no sequence similarity with proteins recorded in gene and protein sequence data banks. A 90-kDa cytosolic CGBG exists in bovine kidneys and reacts with antibodies against CGBG. Binding of ouabain to the cardiac glycoside-binding globulin was monitored by quenching of intrinsic tryptophan fluorescence. Such studies reveal two negatively cooperative ouabain binding sites with Kd' of 1.52 nM and Kd' = 75 nM and with an interaction factor of 50 using a Koshland-Némethy-Filmer model. The demonstration of a cardiac glycoside-binding globulin in plasma is consistent with the recent finding of endogenous cardiac glycosides in mammals.
Antolovic
R
R
Institute of Biochemistry and Endocrinology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Strasse 100, D-35392 Giessen, Germany.
Kost
H
H
Mohadjerani
M
M
Linder
D
D
Linder
M
M
Schoner
W
W
D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002301:Cardiac Glycosides / Q000097:blood*; D002417:Cattle; D002846:Chromatography, Affinity; D004076:Digoxigenin / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010042:Ouabain / Q000494:pharmacology; D012712:Serum Globulins / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013388:Succinimides / Q000378:metabolism
D000345:Affinity Labels; D002301:Cardiac Glycosides; D012712:Serum Globulins; D013388:Succinimides; C028996:cardiac glycoside receptors; C073892:N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate; D010042:Ouabain; D008623:Mercaptoethanol; D000254:Sodium-Potassium-Exchanging ATPase; D004076:Digoxigenin