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

A. Mills, C. D. Demoliou‐Mason, E. A. Barnard

Research output: Contribution to journalArticlepeer-review

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 <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 > 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.

Original languageEnglish
Pages (from-to)904-911
Number of pages8
JournalJournal of Neurochemistry
Volume50
Issue number3
DOIs
Publication statusPublished - 1988

Keywords

  • Bovine cortex
  • Cation and guanine nucleotide effects
  • Detergent solubilization.
  • Neurotensin binding sites

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