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Photolabeling of a 150 kda sodium plus potassium plus chlorine cotransport protein from dog kidney with a bumetanide analogue



Photolabeling of a 150 kda sodium plus potassium plus chlorine cotransport protein from dog kidney with a bumetanide analogue



American Journal of Physiology 253(2 PART 1): C243-C252



(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, [3H]4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid ([3H]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 [3]BSTBA into this protein is completely blocked by inclusion of 10 .mu.M unlabeled bumetanide in the photolysis medium. 2) Photoincorporation of [3H]BSTBA into this protein shows a saturable dependence on [3H]BSTBA concentration, with a K1/2 (.apprx. 0.1 .mu.M) very similar to that for reversible [3H]BSTBA binding to kidney membranes. 3) Photolabeling of this protein by [3H]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 [3H]bumetanide binding and photoincorporation of [3H]BSTBA in the 150-kDa region show a very similar distribution among the 15 gradient fractions collected. [3H]BSTBA is also photoincorporated into at least two lower molecular mass proteins, the largest of which is .apprx. 50 kDa. However, [3H]BSTBA photolabeling of these proteins increases linearly with increasing [3H]BSTBA concentration up to 1.2 .mu.M and is only minimally inhibited when 10 .mu.M unlabeled bumetanide is present during photolysis, suggesting that they are not part of the (Na + K + Cl) cotransporter, or at least that conformation to which bumetanide binds with high affinity. Furthermore, the distribution of [3H]BSTBA photolabeling of these proteins in the sucrose gradient fractions is much different from that of the 150 kDa protein, which is specifically photolabeled by [3H]BSTBA in a manner highly consistent with the (Na + K + Cl) cotransporter.

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Accession: 006110907

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