BINDING OF [I-125] ENDOTHELIN-1 TO RAT CEREBELLAR HOMOGENATES AND ITS INTERACTIONS WITH SOME ANALOGS

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 x 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, κ(+1) = 8.0 ± 1.3 x 105 M-1 s-1; κ(-1) = 2.6 x 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 14 h. 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. 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 Binding was also inhibited by analogues in which either one or both of the cystine disulphide bridges had been replaced by substitution with 2 or 4 alanine residues.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, [Ala1,3,11,15]endothelin-1, was equipotent with endothelin-1 at inhibiting the binding of [125I]-endothelin-1. [Ala3,11]endothelin-1 and [Ala1,15]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.