Studies on ouabain-like endogenous natriuretic factors in human urine. Inhibition of Na-K-ATPase and3H-Ouabain binding

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Abstract
An endogenous inhibitor of sodium transport and of the Na-K-ATPase enzyme was previously detected in the small molecular weight postsalt fraction SIV of serum from saline-loaded rats after gel filtration on Sephadex G-25. In addition, a natriuretic factor present in this fraction of urine from salt-loaded subjects was found to bind to a specific digoxin antibody. Therefore, in the present study the small molecular weight natriuretic and digoxin antibody-binding activities present in the urine of salt-loaded healthy volunteers were purified by reverse-phase chromatography and by immunopřecipitation with the digoxin antibody and were studied for their in vitro effects on Na-K-ATPase derived from hog cerebral cortex. Na-K-ATPase inhibitory activities were found to roughly parallel the natriuretic activities at the various stages of purification. After reverse-phase chromatography, the material of fraction SIV which was bound to the digoxin antibody revealed the highest specific natriuretic activity of 3.95 ± 0.29 µmol/min × mg injected material and showed strongest inhibition of the enzyme with I50 at a concentration of 0.08 µg/ml, as compared with 2.4 µg/ml of the original postsalt urine fraction SIV. Fraction SIV revealed a noncompetitive inhibition of the enzyme with respect to potassium, but also significantly inhibited3H-ouabain binding to the enzyme. Thus, the natriuretic substance(s) present in the urine of salt-loaded healthy subjects exhibit a potent inhibitory effect on the Na-K-ATPase enzyme which is similar, but not identical to that of ouabain.