Synthesis and biological properties of a biotinylated derivative of ACTH1-17 for MSH receptor studies.
A biotinylated derivative of [beta-Ala1,Lys17]-ACTH1-17-NH-(CH2)4-NH2 (ACTH1-17) was synthesized and biologically characterized. The heptadecapeptide with free N-terminus and blocked side-chains was prepared by the solid-phase method using TentaGel resin and a 4-aminobutylamide linker. Biotinyl-beta-Ala-OH was then coupled to the terminal amino group and the resulting [N alpha-(biotinyl-beta-alanyl)-beta-Ala1,Lys17]-ACTH1-17-NH-(CH2)4-N H2 (Bio-ACTH1-17) cleaved from the resin, purified and analyzed. Competition binding assays with mouse B16-F1 and human D10 and HBL melanoma cells using [125I]-alpha-MSH as radioligand gave dissociation constants for Bio-ACTH1-17 of 1.67 +/- 0.07 nM (B16-F1), 0.02 +/- 0.005 nM (D10) and 0.21 +/- 0.02 nM (HBL). The EC50 for Bio-ACTH1-17 in the B16 melanin assay was 4.15 +/- 1.0 nM. Analysis of the binding characteristics of [125I]-Bio-ACTH1-17 demonstrated that in human melanoma cells this radioligand was displaced by ACTH1-17 as well as alpha-MSH whereas in B16-F1 cells the tracer was only displaced from the binding site by ACTH1-17. Studies of Bio-ACTH1-17 with streptavidin showed that the peptide is to a large extent trapped specifically through reaction with biotin. These results demonstrate that (1) the biological characteristics of Bio-ACTH1-17 are almost identical to those of ACTH1-17, (2) Bio-ACTH1-17 is bound by avidin, and (3) Bio-ACTH1-17 may become a useful tool for MSH receptor targeting.
Department of Research (ZLF), University Hospital, Basel, Switzerland.
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D000324:Adrenocorticotropic Hormone / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D001710:Biotin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008543:Melanins / Q000096:biosynthesis; D008546:Melanoma, Experimental / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D011979:Receptors, Pituitary Hormone / Q000378:metabolism*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
C080266:ACTH (1-17)-NH-(CH2)4-NH2, (Nalpha-(biotinyl-beta-Ala(1))-Lys(17))-; D008543:Melanins; D010446:Peptide Fragments; D011979:Receptors, Pituitary Hormone; D001710:Biotin; C029446:MSH receptor; D000324:Adrenocorticotropic Hormone