MONOIODO‐Trp¹¹‐NEUROTENSIN, A NEW LIGAND TO STUDY THE INTERACTION OF NEUROTENSIN WITH ITS RECEPTOR*

Abstract

This work deals with the preparation and binding properties to rat brain synaptic membranes of a biologically active derivative of neurotensin that can be labeled at a specific radioactivity of 2,000 Cilmmol. [zH]neurotensin is the only radioactive ligand of the neurotensin receptor that has been prepared in a pure state. However the specific radioactivity of this tritiated derivative is only 40 to 60 Cil mmol. Because the number of neurotensin binding sites is small in brain and peripheral tissues, there is a need for a biologically active derivative of neurotensin with a much higher specific radioactivity. Iodination of neurotensin gives a complex mixture of peptides in which both Tyr, and Tyr,, have incorporated 1 or 2 iodine atoms per residue. Moreover, we have found that iodination of Tyr,, in acetyl n e u r o t e n~i n~-'~ , a fully active analogue of neurotensin, decreased the biological activity of the hexapeptide by a factor of 20.Trpll-neurotensin, a neurotensin analogue in which Tyr" is substituted by a Trp, was synthesized by the Menifield solid-phase procedure. The biological activity of this analogue was found to be almost identical to that of native neurotensin in an in vitro bioassay involving rat ileum. Iodination of Tyr? in Trp"neurotensin by the lactoperoxidase method was carried out in the presence of nonradioactive [ Y ] N a , containing tracer amounts of radioactive [ 12511]Na. The resulting mixture of diiodo, monoiodo, and unmodified Trp"-neurotensin was purified in one step by ion-exchange chromatography at pH 8.6. The mono and diiodo derivatives of Trp"-neurotensin were pure as judged by UV spectrophotometry, calculation of the number of iodine atoms per mole of peptide, HPLC, and amino-acid analysis. The activity of rnonoiodo Trp'l-neurotensin in the rat ileum bioassay was very similar to that of the unmodified peptide. The use of [ 12"I]Na as the sole source of iodine in the lactoperoxidase-catalyzed iodination reaction allowed to prepare [ 12~I]m~n~iodo-Trp11-ne~r~ten~in with a specific radioactivity of 2,000 Cil mmol. Binding of this analogue to synaptic membranes from rat brain was specific and reversible. The binding isotherm was biphasic and