Subcellular Demonstration of Peroxidatic Oxidation of Tyrosine to Melanin Using Dihydroxyfumarate as Cofactor in Mouse Melanoma Cells

Peroxidatic oxidation of tyrosine was directly demonstrated in tumor cells of Harding Passey mouse melanoma using dihydroxyfumarate (DHF) as cofactor: there was no evidence of oxidation of tyrosine by aerobic dopa oxidase (“tyrosinase”) in these cells. Peroxidative oxidation of tyrosine was correlated with a positive benzidine reaction and peroxidase-dependent oxidation of dopa to melanin. Melanoma cells with peroxidase activity were designated as type A cells.Many of the type A cells also demonstrated oxidation of dopa to melanin which was mediated by aerobic dopa oxidase (“tyrosinase”). In cells with both enzymes, peroxidase activity was present in melanosomes, whereas aerobic dopa oxidase activity was present in both melanosomes and in the Golgi-associated cisternae of endoplasmic reticulum (GERL).Without inhibitors, cells containing both enzymes showed reaction product in melanosomes and in the GERL after incubation in tyrosine, DHF, and hydrogen peroxide. After preincubation with sodium diethyldithiocarbamate (DDC), new melanin was still formed in melanosomes, but was eliminated in the GERL; when catalase was present in the incubation medium, reaction product was completely eliminated in all areas. These results indicate that melanin in the GERL induced after incubation with tyrosine, DHF, and hydrogen peroxide was due to the action of dopa oxidase on dopa synthesized by peroxidase.Some larger, more heavily melanized tumor cells (type B cells) were present which had aerobic dopa oxidase activity, but no demonstrable peroxidase activity. These may represent end-stage cells in which aerobic dopa oxidase acts on dopa formed by previous peroxidase catalysis.Synergism of peroxidase and dopa oxidase may be a mechanism of melanogenesis in vivo