Sensitive Assay and Kinetic Property of urinary Inhibitor of Na+,K+-ATPase in Sodium-loaded Patients with Essential Hypertension : THE 14th CONFERENCE ON THE PATHOGENESIS OF HYPERTENSION

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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 × 10^<-8> M, n=8) under the condition of 100 mM NaCl, 4.5 mM MgSO_4 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℃. 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.
社団法人日本循環器学会の論文
1985-09-20