Photoaffinity labelling of a 150 kDa (Na + K + Cl)-cotransport protein from duck red cells with an analog of bumetanide.
We have used a radiolabelled, benzophenone analog of bumetanide, 4-[3H]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid ([3H]BSTBA) to photolabel plasma membranes from duck red blood cells. BSTBA, like bumetanide, is a loop diuretic and a potent inhibitor of (Na + K + Cl) cotransport, and [3H]BSTBA binds to intact duck red cells with a high affinity similar to that of [3H]bumetanide (K 1/2 congruent to 0.1 microM). We incubated duck red cells with [3H]BSTBA, then lysed the cells and exposed the ghosts to ultraviolet light. The ghosting and photolysis was done at 0 degree C to prevent dissociation of the [3H]BSTBA. The ghosts were then sonicated to remove the nuclei and run on SDS-polyacrylamide gels. Analysis of H2O2-digested gel slices revealed [3H]BSTBA to be incorporated into a protein of approx. 150 kDa. This is the same molecular weight we obtain for a protein from dog kidney membranes which is photolabelled by [3H]BSTBA in a manner highly consistent with labelling of the (Na + K + Cl) cotransporter (Haas and Forbush (1987) Am. J. Physiol. 253, C243-C252). Several lines of evidence strongly suggest that the 150 kDa protein from duck red cell membranes is an integral component of the (Na + K + Cl)-cotransport system in these cells: (1) Photolabelling of this protein by [3H]BSTBA is blocked when 10 microM unlabelled bumetanide is included in the initial incubation medium with [3H]BSTBA; (2) Photoincorporation of [3H]BSTBA into the 150 kDa protein is markedly increased when the initial incubation medium is hypertonic or contains norepinephrine, conditions which similarly stimulate both (Na + K + Cl) cotransport and saturable [3H]bumetanide binding in duck red cells; (3) The photolabelling of this protein shows a saturable dependence on [3H]BSTBA concentration, with a K1/2 (0.06 microM) similar to that for the reversible, saturable binding of [3H]BSTBA and [3H]bumetanide to duck red cells; and (4) [3H]BSTBA photoincorporation into the 150 kDa protein, like saturable [3H]bumetanide binding to intact cells, requires the simultaneous presence of Na+, K+, and Cl- in the medium containing the radiolabelled diuretic.
Haas
M
M
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510.
Forbush
B
B
D000345:Affinity Labels / Q000032:analysis*; D000818:Animals; D001577:Benzophenones / Q000032:analysis* / Q000494:pharmacology; D002034:Bumetanide / Q000378:metabolism; D002352:Carrier Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors; D002712:Chlorides / Q000378:metabolism; D004285:Dogs; D004372:Ducks / Q000097:blood; D004910:Erythrocyte Membrane / Q000032:analysis*; D007668:Kidney / Q000032:analysis; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters; D013424:Sulfanilamides / Q000032:analysis / Q000494:pharmacology
D000345:Affinity Labels; D001577:Benzophenones; D002352:Carrier Proteins; D002712:Chlorides; D028021:Sodium-Potassium-Chloride Symporters; D013424:Sulfanilamides; D002034:Bumetanide; C055204:4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid; D012964:Sodium; D011188:Potassium