Radioreceptor Assay for α;-Msh Using Mouse B16 Melanoma Cells

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Abstract
A radioreceptor assay for α;-MSH is described which is based on cultured mouse B16 melanoma cells and bioactive monoiodinated [Nle4]-α;-MSH tracer. The assay was used (1) to study the binding characteristics of α;-MSH to B16 cells, (2) to determine the relative binding activity of MSH peptides, and (3) to measure MSH in tissue extracts. The association of α;-MSH to B16 cells reached a stable plateau after 3 h at 15°C. At 25° or 37°C, the binding was transient and at 0-1°C, the association was very slow. The hormone-receptor complex was relatively stable between 0° and 15°C whereas a 50% dissociation was reached after 90 min at 25°C and after 35 min at 37°C. The mean KD for α;-MSH of four saturation experiments was 1.3 nM and the number of receptors 9570 per cell. 1,10-Phenanthroline had a stabilizing effect in the binding assay when used at a 0.3 mM concentration. From the MSH peptides tested in the binding assay, some showed similar potencies in three bioassays (tyrosinase, melanin and Anolis skin), whereas others displayed considerably

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