Rates of dissociation of enzyme-ouabain complexes and K_0.5 values in (Na⁺ + K⁺) adenosine triphosphatase from different species

The half-lives of rabbit kidney and dog brain (Na⁺ + K⁺) adenosine triphosphatase (ATPase)-ouabain complexes were 10 and 89 min respectively. The ratios between these half-lives and those previously reported for guinea-pig kidney and rat brain enzyme-ouabain are more than sufficient to account for the differences in the K_0.5 values for ouabain and those enzymes reported in the literature. The K_0.5 values for the ouabain-enzyme interaction could be reduced up to 12-fold by pre-incubating the enzyme with Na⁺, Mg²⁺, ATP and ouabain prior to the addition of K⁺ to start the enzyme assay. These differences show that the K_0.5 values are not obtained under equilibrium conditions and suggest that the true equilibrium position is intermediate between the measured K_0.5 values. K_i values estimated from the dissociation rates of enzyme-ouabain fall between the measured K_0.5 values. It appears that the differences in the dissociation rates alone are sufficient to account for the observed differences in the K_0.5 values. The experiments also show a marked difference in glycoside sensitivity between rat brain and heart (Na⁺ + K⁺) ATPase.