Solubilization and partial purification of the thiazide diuretic receptor from rabbit renal cortex.
This study was designed to solubilize, characterize and begin to purify the thiazide-sensitive Na/Cl transporter from mammalian kidney. Metolazone, a thiazide-like diuretic drug, binds to receptors in rat renal cortex closely related to the thiazide-sensitive Na/Cl transport pathway of the renal distal tubule. In the current study, [3H]metolazone bound to receptors in rabbit renal cortical microsomes. The portion of [3H]metolazone binding that was inhibited by hydrochlorothiazide reflected binding to a high-affinity class of receptor. The affinity (Kd 2.0 +/- 0.1 nM) and number (Bmax = 0.9 +/- 0.4 pmol/mg protein) of high-affinity receptors in rabbit renal cortical membranes were similar to values reported previously for rat. When proximal and distal tubule fragments were separated by Percoll gradient centrifugation, receptors were restricted to the fraction that contained distal tubules. When compared with cortical homogenates, receptor density was enriched 12-fold by magnesium precipitation and differential centrifugation. The zwitterionic detergent CHAPS solubilized 25-35% of the receptors (at 6 mM). Chloride inhibited and Na stimulated binding of [3H]metolazone to solubilized high-affinity receptors. The receptors could be purified significantly by hydroxyapatite chromatography and size exclusion high performance liquid chromatography (HPLC). The combination of magnesium precipitation and differential centrifugation, hydroxyapatite chromatography, and size exclusion HPLC resulted in a 213-fold enrichment of receptors, compared to renal cortical homogenate. The current results indicate that thiazide receptors from rabbit kidney share characteristics with receptors from rat, and that rabbit receptors can be solubilized in CHAPS and purified significantly by hydroxyapatite chromatography and size exclusion HPLC.
West Haven VAMC-Yale University, Center for Renal Research, CT.
D H
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Ellison
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Morrisey
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Desir
D000818:Animals; D001581:Benzothiadiazines; D002352:Carrier Proteins; D002793:Cholic Acids; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004232:Diuretics; D007672:Kidney Cortex / Q000737:chemistry* / Q000378:metabolism; D008788:Metolazone / Q000378:metabolism; D008861:Microsomes / Q000737:chemistry; D011817:Rabbits; D011955:Receptors, Drug / Q000302:isolation & purification*; D049993:Sodium Chloride Symporter Inhibitors / Q000737:chemistry*; D049992:Sodium Chloride Symporters; D012995:Solubility; D027981:Symporters
D001581:Benzothiadiazines; D002352:Carrier Proteins; D002793:Cholic Acids; D004232:Diuretics; D011955:Receptors, Drug; D049993:Sodium Chloride Symporter Inhibitors; D049992:Sodium Chloride Symporters; D027981:Symporters; C064415:thiazide receptor; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D008788:Metolazone