Sensitive assay and kinetic property of urinary inhibitor of Na+, K+-ATPase in sodium-loaded patients with essential hypertension

A sensitive assay method to evaluate the inhibitor of Na+, K+-ATPase in human urine was developed by measuring the inorganic phosphate liberated from ATP in vitro using Na+, K+-ATPase from porcine cerebral cortex. Ouabain inhibited the Na+, K+-ATPase by competing with the potassium ion (an apparent Ki = 2.6 +/- 0.89 X 10(-8) M, n = 8) under the condition of 100 mM NaCl, 4.5 mM MgSO4 and 0.56 mM ATP. The apparent Km value of KCl was 0.4 mM. Factors inhibiting Na+, K+-ATPase were detected in the post-salt fraction on Sephadex G-15 chromatography following the ethanol extraction of lyophilized fresh urine of sodium loaded human subjects (300 meq Na+/day, for 4 days) with essential hypertension. Two active fractions around the 400 daltons following salt were eluted on Sephadex G-15 chromatography. The slower eluted factor competed kinetically with potassium ion, but the inhibitory activity was lost within two days during storage at 4 degrees C. The faster-eluted inhibitor lost its activity within a day. These results indicate that the unstable inhibiting factors of Na+, K+-ATPase exist in human urine and one of these factors inhibits ouabain sensitive Na+, K+-ATPase by binding to the potassium binding site (or very close to it), which exists at the outer surface of the cell membrane of this enzyme.