A protein (Mr = 63,000) from calf serum that promotes the pigmentation of cultured chick neural crest and mouse melanoma cells has been partially isolated and characterized in this study. The stimulation of melanin synthesis in cultured cells was used to follow its activity during purification. The pigment-promoting factor was isolated by sequential column chromatography on dye-agarose matrices followed by hydroxyapatite and high pressure molecular sieve chromatography. The factor was found to stimulate melanin biosynthesis at 2-4 micrograms/ml and was specific for melanin-producing cells and their precursors. Antibodies raised in rabbits against the factor inhibited its pigment-promoting activity as well as that of whole calf serum. Enzyme-linked immunoadsorbent assays demonstrated that calf and bovine sera contain molecules that cross-react with the pigment-promoting factor. Horse, human, rat, and chicken sera, which lack the biological activity, also lacked immunological cross-reactivity. Extracts of certain tissues, particularly the submaxillary gland, were observed to be rich sources of pigment-promoting activity.

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