Photolabeling of a 150-kDa (Na + K + Cl) cotransport protein from dog kidney with a bumetanide analogue
(Na + K + Cl) cotransport is the major mechanism of salt transport across the apical membrane of the epithelial cells of the thick ascending limb of Henle's loop of mammalian kidney and the site of action of loop diuretics such as furosemide and bumetanide. We have identified a 150-kDa protein in membranes from dog kidney cortex that is photolabeled by a radiolabeled, benzophenone analogue of bumetanide, (/sup 3/H)4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid ((/sup 3/H)BSTBA). Several pieces of evidence strongly suggest that this 150-kDa protein is at least part of the (Na + K + Cl) cotransport system. 1) Photoincorporation of (/sup 3/H)BSTBA into this protein is completely blocked by inclusion of 10 microM unlabeled bumetanide in the photolysis medium. 2) Photoincorporation of (/sup 3/H)BSTBA into this protein shows a saturable dependence on (/sup 3/H)BSTBA concentration, with a K 1/2 (approximately 0.1 microM) very similar to that for reversible (/sup 3/H)BSTBA binding to kidney membranes. 3) Photolabeling of this protein by (/sup 3/H)BSTBA requires the simultaneous presence of Na, K, and Cl in the photolysis medium. 4) When crude membranes from dog kidney cortex are centrifuged on sucrose density gradients, saturable (/sup 3/H)bumetanide binding and photoincorporation of (/sup 3/H)BSTBA in the 150-kDa region show a very similar distribution among the 15 gradient fractions collected. (/sup 3/H)BSTBA is also photoincorporated into at least two lower molecular mass proteins, the largest of which is approximately 50 kDa.
- Research Organization:
- Yale Univ., New Haven, CT
- OSTI ID:
- 6243980
- Journal Information:
- Am. J. Physiol.; (United States), Vol. 253
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
CHLORINE COMPOUNDS
MEMBRANE TRANSPORT
POTASSIUM COMPOUNDS
PROTEINS
LABELLING
SODIUM COMPOUNDS
BENZOIC ACID
CELL MEMBRANES
DOGS
EPITHELIUM
KIDNEYS
MOLECULAR WEIGHT
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ALKALI METAL COMPOUNDS
ANIMAL TISSUES
ANIMALS
BODY
CARBOXYLIC ACIDS
CELL CONSTITUENTS
HALOGEN COMPOUNDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MAMMALS
MEMBRANES
MONOCARBOXYLIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
TISSUES
VERTEBRATES
551001* - Physiological Systems- Tracer Techniques