125I-(Thr34, Nle37)- CCK31–39A Non Oxidizable Tracer for the Characterization of CCK Receptor on Pancreatic Acini and Radio-Immunoassay of C-Terminal CCK Peptides

Abstract

A derivative of the C-terminal nonapeptide of cholecystokinin, i.e., (Thr34, Nle37) [34- threonine, 37-norleucine]- CCK31-39 was radioiodinated by conjugation with 125I-Bolton-Hunter reagent. The labeled peptide was purified by RP-HPLC [reverse-phase high pressure liquid chromatography] on a C-18 column. Validation of the iodinated peptide was performed by measuring its biological integrity and by studying its binding characteristics on pancreatic acini [guinea-pig]. 125I-(Thr, Nle)-CCK-9 present the same ability to stimulate amylase release as (Thr, Nle)- CCK-9 and CCK-8. Binding of the radioligand to CCK receptors is specific, reversible and saturable. Inhibition of the binding by CCK-related peptides correlates well with their biological potencies. 125I-(Thr, Nle)-CCK-9 is able to interact with high affinity CCK receptors. Furthermore, 125I-(Thr, Nle)-CCK-9 is recognized by C-terminal CCK directed antibodies. This reliable tracer could be used as a replacement of CCK-8 since it is protected from risks of oxidation.