The K+-dependent phosphatase of rat kidney. Its properties and the effects of maneuvers that modify (Na+ + K+)-ATPase activity.

The properties of rat renal p-nitrophenylphosphatase have been investigated and the effects of certain experimental maneuvers (known to modify renal (Na+ + K+)-ATPase) on p-nitrophenylphosphatase specific activity have been examined. The rat kidney enzyme had an optimal pH in the range 7–8 and the temperature dependence of enzyme activity (Arrhenius plot) yielded a single slope with an activation energy of 40 kJ/mol. (Na+ + K+)-ATPase assayed in the same enzyme preparation yielded a biphasic plot with activation energies of 81 and 160 kJ/mol and phase transition at 20°C. The Km value for p-nitrophenyl phosphate was 1.62 mM and the K12 value for potassium was 2.2–3.3 mM. ATP had a dual inhibitory effect on the potassium activation of the phosphatase: at low concentrations a non-competitive pattern was observed (Ki = 0.015 mM), and at high concentrations a typical competitive inhibitory pattern occurred (Ki = 0.04 mM). Sodium at low concentrations produced a substantial decrease in the affinity of the phosphatase for potassium (1.8 to 6mM); higher concentrations of sodium did not have additional effects on the potassium affinity but markedly reduced the V value of the reaction. Ouabain (1–5mM) inhibited basal enzyme activity by 80%. Preincubation of the enzyme with ouabain produced mild stimulation at low concentrations of the glycoside (1–100 μM) and resulted in 100% inhibition of basal activity at high concentrations (1–5 mM). Ouabain inhibited p-nitrophenyl phosphate activation of the phosphatase by a pattern similar to that observed with sodium: a decrease in the apparent affinity for p-nitrophenyl phosphate (1.62 to 3 mM) at low concentrations of the glycoside and a decrease in V at higher concentrations without additional effects on the affinity. Ouabain inhibition of the potassium activation of the phosphatase displayed kinetics of uncompetitive inhibition. Unlike the enzyme from red blood cells, calcium, in the presence of ATP, did not result in loss of ouabain sensitivity. The specific activities of both the potassium-activated and ouabain-sensitive phosphatases changed in parallel with changes in (Na+ + K+)-ATPase under the following experimental conditions: adrenalectomy, chronic dexamethasone treatment, potassium loading, acute dexamethasone treatment and reduction of renal mass. The changes in the specific activities of the two enzymes were quantitatively correlated in the cortex and medulla following adrenalectomy and chronic dexamethasone treatment. A similar correlation was found in the cortex (but not in the medulla) after potassium loading. Stimulation by acute dexamethasone treatment of p-nitrophenylphosphatase in the renal cortex (but not in the medulla) was substantially larger than the corresponding stimulation of (Na+ + K+)-ATPase. After reduction of renal mass, stimulation of (Na+ + K+)-ATPase was not consistently observed. When it occurred, it was confined to the renal medulla and it was accompanied by increases in the specific activity of p-nitrophenylphosphatase. But the magnitude of p-nitrophenylphosphatase stimulation was substantially lower than that of (Na+ + K+)-ATPase. These observations and the quantitative aspects of the correlations between renal (Na+ + K+)-ATPase and p-nitrophenylphosphatase suggest that under all the experimental conditions examined, (Na+ + K+)-ATPase activation may reflect an increase in the number of enzyme units. In addition, the results suggest that in certain situations (acute dexamethasone treatment, reduction of renal mass), additional effects on the kinetic properties of the transport enzyme may also occur.