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 100kDa polypeptide. K(m) for Pi was 17.7+/-1.82 microM and 16.8+/-0.69 microM for ouabain-induced and furosemide-induced phosphorylation, respectively. K(m) 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 K(m) 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.
Thomas
Luz E
LE
Laboratorio de Fisiología Gastrointestinal, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas (IVIC), Apartado 21827, 1020-A Caracas, Venezuela.
Burguillos
Luis
L
del Castillo
Jesús R
JR
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D020895:Enterocytes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D005665:Furosemide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007421:Intestine, Small / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000145:classification* / Q000378:metabolism*; D013552:Swine
D010750:Phosphoproteins; D010042:Ouabain; D005665:Furosemide; D000254:Sodium-Potassium-Exchanging ATPase