Synthesis and iodination of human (phenylalanine 13, tyrosine 19) melanin-concentrating hormone for radioreceptor assay.
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 Fmoc-strategy 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 x 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 alpha-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.
Drozdz
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Department of Research (ZLF), University Hospital, Basel, Switzerland.
Eberle
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D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D007028:Hypothalamic Hormones / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D008543:Melanins / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D008545:Melanoma / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010907:Pituitary Hormones / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D011869:Radioligand Assay / Q000379:methods*; D011979:Receptors, Pituitary Hormone / Q000378:metabolism; D014407:Tumor Cells, Cultured
D007028:Hypothalamic Hormones; D007457:Iodine Radioisotopes; D008543:Melanins; D010907:Pituitary Hormones; D011979:Receptors, Pituitary Hormone; C092274:melanin-concentrating hormone receptor; C019463:melanin-concentrating hormone