Isolation and reconstitution of furosemide-binding proteins from Ehrlich ascites tumor cells.
Furosemide-binding proteins were isolated from cholate-solubilized membranes of Ehrlich ascites tumor cells by affinity chromatography, using furosemide as ligand. Solubilized proteins retarded by the affinity material were eluted by furosemide. In reducing and denaturing gels, the major proteins eluted by furosemide were 100 and 45 kDa. In nonreducing, non-denaturing gels, homodimers of both polypeptides were found, whereas no oligomeric proteins containing both polypeptides were seen. It is concluded that the furosemide gel binds two distinct dimeric proteins. The isolated proteins were reconstituted into phospholipid vesicles and the K+ transport activity of these vesicles was assayed by measurement of 86Rb+ uptake against a large opposing K+ gradient. The reconstituted system was found to contain a K+ transporting protein, which is sensitive to Ba2+ like the K+ channel previously demonstrated to be activated in intact cells after cell swelling.
Jessen
F
F
Institute of Biological Chemistry A, August Krogh Institute, University of Copenhagen, Denmark.
Cherksey
B D
BD
Zeuthen
T
T
Hoffmann
E K
EK
D000818:Animals; D000837:Anion Exchange Resins / Q000378:metabolism; D001464:Barium; D002034:Bumetanide; D002286:Carcinoma, Ehrlich Tumor / Q000032:analysis / Q000473:pathology*; D002712:Chlorides / Q000493:pharmacokinetics; D002846:Chromatography, Affinity; D003300:Copper; D004591:Electrophoresis, Polyacrylamide Gel; D005665:Furosemide / Q000378:metabolism* / Q000494:pharmacology; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009363:Neoplasm Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D010618:Phenanthrolines; D011188:Potassium / Q000493:pharmacokinetics; D011803:Quinine; D012413:Rubidium / Q000493:pharmacokinetics; D014407:Tumor Cells, Cultured
D000837:Anion Exchange Resins; D002712:Chlorides; D007473:Ion Channels; D009363:Neoplasm Proteins; D010455:Peptides; D010618:Phenanthrolines; D002034:Bumetanide; D001464:Barium; D003300:Copper; D005665:Furosemide; D011803:Quinine; D012413:Rubidium; D011188:Potassium