Digitalis-like activity in human plasma. Purification, affinity, and mechanism

• Published in: The Journal of biological chemistry Vol. 264; no. 13; pp. 7395 - 7404
• Main Authors: HAMLYN, J. M, HARRIS, D. W, LUDENS, J. H
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05-05-1989
• Subjects: Analytical, structural and metabolic biochemistry; Binding, Competitive; Biological and medical sciences; Ca(2+) Mg(2+)-ATPase - antagonists & inhibitors; Calcium-Transporting ATPases - antagonists & inhibitors; Chromatography, High Pressure Liquid; Enzyme Inhibitors - blood; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Humans; Hydrolases; In Vitro Techniques; Ligands; man; Molecular Weight; Ouabain - pharmacology; Phosphorylation; plasma; Sodium-Potassium-Exchanging ATPase - antagonists & inhibitors; Human; Purification; Affinity chromatography; Enzyme; Na,K-ATPase; HPLC chromatography; Molecular interaction; Endogenous digitalis-like factor; Blood plasma
• ISSN: 0021-9258; 1083-351X

A factor having digitalis-like characteristics has been isolated from human plasma and its mechanism of action compared with
the commonly used cardenolide, ouabain. The purification scheme involved dialysis of human plasma, lyophilization of dialysate,
extraction of methanol-soluble components, and flash evaporation, followed by preparative, semipreparative, and analytical
scale reverse-phase chromatography. One peak of biologically active material was obtained and shown to possess digitalis-like
activity in assays of sodium pump activity, receptor binding, and Na,K-ATPase activity. Results from (i) the determination
of the ligand conditions supporting binding, (ii) kinetics of association and dissociation from the Na,K-ATPase, (iii) affinity
titration, (iv) selectivity, and (v) competition studies, when taken together, show that the endogenous digitalis-like factor
is a specific inhibitor of the sodium pump that stabilizes the E2P form of the enzyme in a manner analogous to ouabain. The
endogenous digitalis-like factor binds competitively in or near the receptor site for cardiac glycosides with an apparent
affinity 8-20-fold greater than any known cardioactive steroid. The presence of digitalis-like activity in the circulation
of individuals with no known intake of these compounds suggests that the material characterized here is an endogenous counterpart
to the cardenolides. This factor may regulate sodium pump activity and provide a rationale for the existence of gene and tissue-specific
forms of the Na,K-ATPase having distinct sensitivity to the cardenolides.