THE MOUSE BROWN (B)-LOCUS PROTEIN FUNCTIONS AS A DOPACHROME TAUTOMERASE

The mouse b locus controls black/brown coat coloration. Its product, t he b-protein or TRP-1, has significant homology to tyrosinase, and thi s has led to suggestions that the b-protein is itself a melanogenic en zyme. In order to investigate its function, we have used lines of mous e fibroblasts stably expressing the b-protein. We were unable to confi rm previous reports that the b-protein has tyrosinase or catalase acti vity, but detected stereospecific dopachrome tautomerase activity in b -protein-expressing fibroblasts. This dopachrome tautomerase binds to Concanavalin A-Sepharose, and the major product of its action on L-dop achrome is 5,6-dihydroxyindole-2-carboxylic acid, as expected for the mammalian enzyme. Since this activity is not present in untransfected fibroblasts we conclude that the b-protein has dopachrome tautomerase activity. Further supporting evidence comes from the analysis of melan in metabolites produced by fibroblasts expressing tyrosinase alone, or in combination with the b-protein. Culture medium from the line expre ssing both proteins contains significant amounts of methylated carboxy lated indoles, such as 6-hydroxy-5-methoxyindole-2-carboxylic acid, wh ich would be expected in cells with an active dopachrome tautomerase. The levels of these compounds in medium from cells expressing tyrosina se alone are approximately 20-fold lower, and not significantly above background. Hence, it appears that the b-protein acts as a dopachrome tautomerase in vivo as well as in vitro.