Phosphorylated intermediate of the ouabain-insensitive,Na-stimulated ATPase in rat kidney cortex and rainbow trout gills

Several tissues from different animals, including the rat kidney and the freshwater rainbow trout gills, show an ouabain-insensitive, furosemide-sensitive, Na⁺-stimulated ATPase activity, which has been associated with the active control of the cell volume. This Na-ATPase is Mg²⁺ dependent and it is inhibited by vanadate, which can be taken as an indication that this enzyme is a P-type ATPase. The P-type ATPases are known to form a phosphorylated intermediate during their catalytic cycle, where the phosphate binds an aspartyl residue at the enzyme's substrate site. In the current study, we partially characterized the phosphorylated intermediate of the ouabain-insensitive Na-ATPase of rat kidney cortex homogenates and that of gill microsomes from freshwater rainbow trout. While the kidney cortex homogenates, under our assay conditions, show both Na- and Na,K-ATPase activities, the gill microsomes, when assayed at pH 5.2, only show Na-ATPase activity. Both preparations showed a Mg²⁺-dependent, Na⁺-stimulated phosphorylated intermediate, which is enhanced by furosemide. Incubation of the phosphorylated enzyme with 0.6 N hydroxylamine (NH₂OH) showed that it is acid-stable and sensitive to hydroxylamine, either when phosphorylated in the presence or absence of furosemide. Addition of ADP to the incubation medium drives the reaction cycle of the enzyme backward, diminishing its phosphorylation. Na⁺ seems to stimulate both the phosphorylation and the dephosphorylation of the enzyme, at least for the Na-ATPase from gill microsomes. In a E1–E2 reaction cycle of the Na-ATPase, furosemide seems to be blocking the transition step from Na·E1∼P to Na·E2-P.