Figure 4. Effect of temperature, pH, and vanadate sensitivity of the CPZ treated enzyme.

A. Pig kidney Na⁺,K⁺-ATPase activity was measured at four different temperatures, as described in Materials and Methods, in the presence of the indicated CPZ concentrations. The reaction contained 30 mM histidine buffer, pH 7.2, 100 mM NaCl, 20 mM KCl, 3 mM MgCl₂, and 3 mM ATP. The inhibition curves were analyzed using the Hill equation, giving the following IC₅₀ values; squares, 5°C (7.64±1.14 µM); circles, 15°C (13.88±1.05 µM); diamonds, 30°C (37.33±1.08 µM); and triangles, 37 °C (108.20±1.31 µM). B. Effect of pH on CPZ inhibition of Na⁺-ATPase activity. Na⁺-ATPase activity was measured as described in panel A, but in the absence of K⁺ and in the presence of different pH values (the reaction was buffered with 30 mM Tris adjusted with HCl), in the presence of the indicated CPZ concentrations. Squares, circles, and diamonds indicate measurements performed at pH 6, 7, or 8, respectively. C. Na⁺,K⁺-ATPase (NKA) activity was measured as in panel A, but in the presence of 1 mM ATP and the indicated vanadate concentrations at 23°C. The ATPase mixture contained either DMSO (squares) or 20 µM CPZ (circles).