POLYMERIZATION OF 5,6-DIHYDROXYINDOLE-2-CARBOXYLIC ACID TO MELANIN BYTHE PMEL-17 SILVER LOCUS PROTEIN

Recent advances in melanogenesis have focused on the role of dihydroxy indole-2-carboxylic acid [(HO)(2)IndCOOH]. For example, it has been sh own that formation of (HO)(2)IndCOOH from dopachrome is catalyzed by d opachrome tautomerase, that the melanogenic protein tyrosinase-related protein (TRP)-1 can oxidize (HO)(2)IndCOOH to its indole quinone, tha t (HO)(2)IndCOOH-melanins can be synthesized chemically, that mammalia n melanins are naturally rich in (HO)(2)IndCOOH subunits, and that (HO )(2)Ind- COOH is incorporated into melanins of melanomas in mice. The question thus emerges as to the mechanism(s) by which (HO)(2)IndCOOH a nd other precursors become incorporated into melanins in vivo. Accordi ngly, an activity was partially purified that catalyzed melanin format ion with (HO)(2)IndCOOH as a substrate. Analyses of the (HO)(2)IndCOOH polymerization factor from Cloudman melanoma cells revealed the follo wing: it was proteinaceous in that it was heat labile and destroyed by proteinase K; it was a glycoprotein in that it adhered to wheat germ agglutinin and was eluted with N-acetyl glucosamine; it was located pr edominantly in the melanosomal fraction of cell homogenates; the activ ity was reduced by exposure to the metal chelators EDTA and EGTA, but not by phenylthiourea, a tyrosinase inhibitor; the (HO)(2)IndCOOH poly merization reaction was inhibited by superoxide dismutase. In addition , the activity was found with the mouse pmel 17/silver locus protein i mmunopurified from human melanoma cells, and was significantly reduced in extracts of mouse melanocytes cultured from silver (si/si) mice co mpared to extracts from Si/Si melanocytes. in summary, an activity has been identified in human and mouse melanoma cells that catalyzes the superoxide-dependent polymerization of (HO)(2)IndCOOH to melanin in vi tro, and appears to be a function of the pmel 17/silver protein of the human pmel 17 gene and the mouse silver locus.