Solubilization of a guanine nucleotide-sensitive parathyroid hormone-receptor complex from canine renal cortex.
Canine renal cortical PTH receptors were solubilized after occupancy of membrane-associated receptors with the agonist ligand [125I]bovine (b) PTH-(1-34). Stabilization of binding during solubilization required the use of high concentrations of BSA (optimally 5%) and appropriate detergents (0.5% 3-[(3-cholamidopropyl)dimethylammonio]2-hydroxy-1-propanesulfonate, 0.5% 3-[(3-cholamidopropanyl)dimethylammonio]1-propanesulfonate, or 0.5-1.0% digitonin). The soluble fraction (240,000 X gav supernatant) contained [125I]bPTH-(1-34) associated with macromolecular components as well as unbound [125I]bPTH-(1-34) that dissociated during solubilization. The soluble macromolecular complex had functional properties expected of a ternary complex consisting of [125I]bPTH-(1-34) receptor stimulatory guanine nucleotide-binding protein (Ns). Thus, the dissociation of labeled PTH at 30 C was slow (t1/2 = 75 min); in the presence of GTP (10(-4) M), 75% of the sites displayed rapid dissociation kinetics (t1/2 = 2.3 min). This effect was nucleotide specific, with GTP approximately equal to GTP gamma S approximately equal to GDP greater than GDP beta S greater than ITP approximately equal to guanylylimidodiphosphate much greater than GMP approximately equal to App(NH)p. ATP was ineffective. GTP produced a half-maximal response at a concentration of 200 nM. These results are consistent with the reported nucleotide specificity and affinity of purified Ns. Treatment of membranes with N-ethylmaleimide during the binding reaction rendered the solubilized complex refractory to GTP. Gel filtration chromatography (Sepharose 6B) revealed a GTP-sensitive complex that eluted in the position expected of a detergent-free spherical protein of 180,000 daltons. This complex may consist of the 60,000 to 70,000-dalton PTH-binding subunit (previously identified by photoaffinity labeling) together with Ns.
D000818:Animals; D002850:Chromatography, Gel; D003902:Detergents; D004285:Dogs; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism; D007672:Kidney Cortex / Q000032:analysis*; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000302:isolation & purification; D010281:Parathyroid Hormone / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D018016:Receptors, Parathyroid Hormone; D012710:Serum Albumin, Bovine; D012995:Solubility
D003902:Detergents; D006150:Guanine Nucleotides; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010281:Parathyroid Hormone; D011956:Receptors, Cell Surface; D018016:Receptors, Parathyroid Hormone; D012710:Serum Albumin, Bovine; D019204:GTP-Binding Proteins; D005033:Ethylmaleimide