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

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Abstract
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 (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 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 (KD= 5.5 nM, Bmax= 250 fmol/mg protein, nH= 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 (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.

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