Synthesis and lodination of human (phenylalanine13, tyrosine19) melanin‐concentrating hormone for radioreceptor assay

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Abstract
An analogue of human melanin‐concentrating hormone (MCH) suitable for radioiodination was designed in which Tyr13 and Val19 of the natural peptide were replaced by phenylalanyl and tyrosyl residues: [Phe13, Tyr19] ‐MCH. The peptide was synthesized by the continuous‐flow solid‐phase methodology using Fmocstrategy and Polyhipe PA 500 and PEG‐PS resins. The linear MCH peptides with either acetamidomethyl‐protected or free cysteinyl residues were purified to homogeneity and cyclized by iodine oxidation, yielding the final product with the correct molecular weight of 2434.61. Radioiodination of the C‐terminal tyrosine was carried out enzymatically using solid‐phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed‐phase mini‐column and by high‐pressure liquid chromatography. The resulting [125I]‐[Phe13, Tyr19]‐MCH tracer was the first radiolabelled MCH peptide suitable for radioreceptor assay: saturation binding analysis using mouse G4F‐7 melanoma cells demonstrated the presence of 1090 MCH receptors per cell. The dissociation constant (KD) was 1.18 × 10−10 M, indicating high‐affinity MCH receptors on these cells. MCH receptors were also found in other cell lines such as mouse B16‐F1 and G4F and human RE melanoma cells as well as in PC12 and COS‐7 cells. Competition binding analyses with a number of other peptides such as α‐MSH, neuropeptide Y, substance P and pituitary adenylate cyclase activating peptide, demonstrated that the binding to the MCH receptor is specific. Atrial natriuretic factor was found to be a weak competitor of MCH, indicating topological similarities between MCH and ANF when interacting with MCH receptors.