Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Analysis of the equilibrium binding of [³H]‐neurotensin(1–13) at 25°C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (K_D) of 3.3 nM and a density (B_max) of 350 fmol/mg protein (Hill coefficient n_H= 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising <10% of the total. K_D 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 > neurotensin(8–13) > 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 (K_D= 5.5 nM, B_max= 250 fmol/mg protein, n_H= 1.0) was obtained in 2% digitonin/l mM Mg²⁺ extracts of membranes which had been preincubated (25°C, 1 h) with 1 mM Mg²⁺ prior to solubilization. Association‐dissociation kinetic studies then revealed the presence of two classes of sites (K_D1= 0.5 nM, K_D2= 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.