title: The mouse brown (b) locus protein has dopachrome tautomerase activity and is located in lysosomes in transfected fibroblasts.
abstract: Many genes mapping to pigmentation loci are involved in the regulation of melanin synthesis in the mouse. The brown (b) locus controls black/brown coat coloration, and its product has significant homology to the key melanogenic enzyme tyrosinase. This has led to suggestions that the b-protein is itself a melanogenic enzyme. In order to investigate its function, we have established lines of mouse fibroblasts stably expressing the b-protein by co-transfection of a b-protein expression vector and a plasmid conferring resistance to the antibiotic G418. The b-protein synthesised by these cells has the expected molecular mass of 75 kDa and reacts with three different anti-b-protein antibodies. We were unable to confirm previous reports that the b-protein has tyrosinase or catalase activity, 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-dopachrome is 5,6-dihydroxyindole-2-carboxylic acid. Since this activity is not present in untransfected cells we conclude that the b-protein has dopachrome tautomerase activity. Fibroblasts do not contain melanosomes, the specialised organelles in which the b-protein is located in melanocytes. Nevertheless, indirect immunofluorescence localisation of the b-protein in transfected fibroblasts produces a distinctive pattern of intense juxtanuclear staining combined with punctate cytoplasmic staining. Double-labelling shows co-localisation of the b-protein with the late endosomal/lysosomal markers beta-glucuronidase and LAMP-1, both in transfected fibroblasts and in mouse melanoma cells. These findings are consistent with the hypothesis that melanosomes are closely related to lysosomes.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, UK.
authors.forename: A J
authors.initials: AJ
authors.lastname: Winder
authors.forename: A
authors.initials: A
authors.lastname: Wittbjer
authors.forename: E
authors.initials: E
authors.lastname: Rosengren
authors.forename: H
authors.initials: H
authors.lastname: Rorsman
mesh_terms: D016475:3T3 Cells / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D002374:Catalase / Q000032:analysis; D016384:Consensus Sequence; D004790:Enzyme Induction; D005347:Fibroblasts / Q000201:enzymology*; D005966:Glucuronidase / Q000032:analysis; D006056:Golgi Apparatus / Q000378:metabolism; D006200:Hair Color / Q000235:genetics*; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000235:genetics*; D008247:Lysosomes / Q000201:enzymology*; D008543:Melanins / Q000096:biosynthesis; D051379:Mice / Q000235:genetics*; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains / Q000235:genetics; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000032:analysis; D010544:Peroxidases / Q000032:analysis; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D008543:Melanins; D010544:Peroxidases; D002374:Catalase; D014442:Monophenol Monooxygenase; D005966:Glucuronidase; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase