Binding of [125I]‐endothelin‐1 to rat cerebellar homogenates and its interactions with some analogues

Abstract

1 [¹²⁵I]-endothelin-1, over the concentration range 6 pm–10 nm, bound to a single site in homogenates of rat cerebellum with high affinity (K_d = 2.8 ± 0.6 × 10⁻¹⁰ m). The site was present in a concentration of 321 ± 58 fmol mg⁻¹ protein. 2 The rates of association and dissociation of [¹²⁵I]-endothelin-1 with the binding site were slow (at 25°C, k₊₁ = 8.0 ± 1.3 × 10⁵m⁻¹ s⁻¹; k₋₁ = 2.6 × 10⁻⁴ s⁻¹) and, on addition of a maximally displacing concentration of endothelin-1 (100 nm), 94.0 ± 8.4% of the [¹²⁵I]-endothelin-1 was still bound after 14h. 3 [¹²⁵I]-endothelin-1 binding was inhibited by a number of naturally occurring or genetically encoded members of the endothelin/sarafotoxin family of peptides. The order of potency was endothelin-3 = sarafotoxin S6b > endothelin-2 = endothelin-1 ≫ porcine proendothelin_1–39. 4 Binding was also inhibited by analogues in which either one or both of the cystine disulphide bridges had been replaced by substitution with 2 or 4 alanine residues. The tetra-alanyl substituted analogue, [Ala^1,3,11,15]endothelin-1, was equipotent with endothelin-1 at inhibiting the binding of [¹²⁵I]-endothelin-1. [Ala^3,11]endothelin-1 and [Ala^1,15]endothelin-1, analogues which each contained one of the disulphide bridges from the parent peptide, were respectively 3 and 14 times less potent than the parent peptide. An analogue in which the Glu¹⁰ residue had been anisylated was 25 fold less potent than endothelin-1. 5 It is concluded that the structural requirements for binding to the cerebellar sites for [¹²⁵I]-endothelin-1 do not require the presence of the disulphide bridges characteristic of the endothelin/sarafotoxin family. Rather, the binding may be more sensitive to the presence of bulky side chain substituents, at least in the smaller intramolecular loop.