Characterization of neurotensin binding sites in intact and solubilized bovine brain membranes.
Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.
Mills
A
A
MRC Molecular Neurobiology Unit, Medical Research Council Centre, Cambridge, England.
Demoliou-Mason
C D
CD
Barnard
E A
EA
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002412:Cations / Q000494:pharmacology; D002417:Cattle; D002540:Cerebral Cortex / Q000378:metabolism*; D003902:Detergents; D004355:Drug Stability; D008566:Membranes / Q000378:metabolism; D009496:Neurotensin / Q000031:analogs & derivatives / Q000378:metabolism*; D012995:Solubility; D014316:Tritium
D002412:Cations; D003902:Detergents; D014316:Tritium; D009496:Neurotensin