FIGURE 9.

Effect of phosphorylation on the inhibition of Na,K-ATPase activity by cardiotonic steroids. Hydrolytic activity (20 °C) of enzyme following preincubation for various time periods with 1 μm ouabain (○), ouabagenin (□), or anthroyl ouabain (△) and either Mg²⁺ alone (5 mm), or Mg²⁺ (5 mm) plus P_i (1 mm). The data were fitted with a monoexponential function. The observed rate constants (k_obs) and relative inhibition of the fits in the presence of only Mg²⁺ (gray curves) were: ouabain, 0.019 ± 0.004 s⁻¹ and 8.5%; OG, 0.025 ± 0.005 and 10.3%; AO, 0.024 ± 0.006 s⁻¹ and 11.0%. In the presence of both Mg²⁺ and P_i (black curves) the fitting parameters were: ouabain, 0.013 ± 0.001 s⁻¹ and 40.0%; OG, 0.022 ± 0.003 s⁻¹, and 24.4%; AO, 0.016 ± 0.002 s⁻¹, and 40.0%.