Solubilization of thiazide diuretic receptors from rat kidney membranes.
Thiazide-type diuretics act at receptors to inhibit NaCl transport in the renal distal tubule. We solubilized high-affinity [3H]metolazone binding sites from rat kidney membranes with Triton X-100, which was more effective than several other detergents. Phosphatidylcholine and a mixture of proteinase inhibitors were needed to stabilize binding so that 57% of solubilized binding remained after 72 h at 4 degrees C. The affinities of solubilized (Kd = 11.4 +/- 0.5 nM) and membrane-bound receptors (Kd = 12.0 +/- 1.7 nM) were similar. The maximal number of binding sites/mg protein of solubilized receptors was 46 +/- 3% (n = 5) of membrane-bound receptors. Diuretics with a wide range of affinities had similar affinities for the solubilized and membrane-bound sites. Chloride inhibited and sodium stimulated the binding of [3H]metolazone to solubilized receptors, as they do with membrane-bound receptors. These studies demonstrate that, as judged by ligand binding, thiazide receptors can be solubilized in an active conformation and provide the basis for future purification and reconstitution.
Luo
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H
Department of Medicine, University of California, San Diego, La Jolla 92093.
Beaumont
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Vaughn
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Fanestil
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D000818:Animals; D001581:Benzothiadiazines; D001665:Binding Sites; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents; D004232:Diuretics / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism*; D007686:Kidney Tubules, Distal / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008788:Metolazone / Q000378:metabolism*; D010713:Phosphatidylcholines; D011480:Protease Inhibitors; D051381:Rats; D011919:Rats, Inbred Strains; D011955:Receptors, Drug / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D049993:Sodium Chloride Symporter Inhibitors / Q000494:pharmacology; D012995:Solubility
D001581:Benzothiadiazines; D003902:Detergents; D004232:Diuretics; D010713:Phosphatidylcholines; D011480:Protease Inhibitors; D011955:Receptors, Drug; D049993:Sodium Chloride Symporter Inhibitors; D008788:Metolazone