Phosphorylation of two isozymes of (Na + + K +)-ATPase by inorganic phosphate

Abstract

The phosphorylation of two isozymes (α(+) and a) of (Na + + K +)-ATPase by 32 P i was studied under equilibrium conditions in various enzyme preparations from rat medulla oblongata, rat cerebral cortex, rat cerebellum, rat kidney, guinea pig kidney, and rabbit kidney. In ouabain-sensitive (Na + + K +)-ATPases such as the brain, guinea pig kidney, and rabbit kidney enzymes, ouabain stimulated the Mg 2+-dependent phosphorylation at lower concentrations, while a higher concentration was required for the stimulation of rat kidney (Na + + K +)-ATPase, an ouabain-insensitive enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that two isozymes of the brain (Na + + K +)-ATPase were also phosphorylated by 32 P i in the presence of ouabain. The properties of the phosphorylation were compared between the medullar oblongata (referred to as α(+)) and the kidney (referred to as α) (Na + + K +)-ATPases. The steady-state level of phosphorylation was achieved faster in the kidney enzymes than in the medulla oblongata enzyme. Phosphorylation without ouabain was greater in the kidney enzymes than in the brain enzymes. Furthermore, the former enzymes were inhibited by K + much more than the latter. These findings suggest that the two isozymes of (Na + + K +)-ATPase differ in their conformational changes during enzyme turnover.