Backdoor phosphorylation of basolateral plasma membranes of small intestinal epithelial cells: characterization of a furosemide-induced phosphoprotein related to the second sodium pump

Enterocyte has two different Na+-stimulated ATPases, the ouabain-sensitive Na+/K+ ATPase and a furosemide-inhibitable Na+ ATPase. To identify the polypeptide associated with the Na+-ATPase, 32Pi phosphorylation into basolateral membranes of enterocyte was investigated. Both, ouabain and furosemide induced Mg2+-dependent, vanadate-sensitive 32Pi incorporation into a 100 kDa polypeptide. Km for Pi was 17.7 ± 1.82 μM and 16.8 ± 0.69 μM for ouabain-induced and furosemide-induced phosphorylation, respectively. Km for furosemide was 1.3 ± 0.21 mM. Furosemide-induced 32Pi incorporation was sensitive to alkaline pH and hydroxylamine suggesting an acyl-phosphate bond. Na+ and K+ inhibited 32Pi incorporation induced by ouabain. In contrast, Na+ stimulated furosemide-induced phosphorylation with a Km of 16.5 ± 5.59 mM while K+ had no effect. Purified Na+/K+ ATPase only presented ouabain-induced phosphoprotein, indicating that furosemide-induced phosphorylation is not related to this enzyme and appears to correspond to a new member of P-type ATPases associated with the second Na+ pump.