Isolation and reconstitution of furosemide-binding proteins from Ehrlich ascites tumor cells

• Published in: The Journal of membrane biology 1989-05, Vol.108 (2), p.139-151
• Main Authors: Jessen, F, Cherksey, B D, Zeuthen, T, Hoffmann, E K
• Format: Article
• Language: English
• Subjects: Animals; Anion Exchange Resins - metabolism; Barium; Bumetanide; Carcinoma, Ehrlich Tumor - analysis; Carcinoma, Ehrlich Tumor - pathology; Chlorides - pharmacokinetics; Chromatography, Affinity; Copper; Ehrlich ascites tumor cells; Electrophoresis, Polyacrylamide Gel; Furosemide - metabolism; Furosemide - pharmacology; Ion Channels - drug effects; Ion Channels - metabolism; membrane proteins; Mice; Neoplasm Proteins - analysis; Neoplasm Proteins - isolation & purification; Neoplasm Proteins - metabolism; Peptides - analysis; Peptides - isolation & purification; Peptides - metabolism; Phenanthrolines; Potassium - pharmacokinetics; Quinine; Rubidium - pharmacokinetics; Tumor Cells, Cultured
• ISSN: 0022-2631; 1432-1424
• DOI: 10.1007/BF01871025

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.