THE DHICA OXIDASE ACTIVITY OF THE MELANOSOMAL TYROSINASES LEMT AND HEMT

Although melanins can be formed in vitro by the unique action of tyros inase on L- tyrosine, it is now well accepted that other enzymes terme d tyrosinase-related proteins are involved in mammalian melanogenesis. However, some aspects of their roles in the regulation of the pathway are still unknown. The action of dopachrome tautomerase on L-dopachro me yields DHICA, a stable dihydroxyindole with a low rate of spontaneo us oxidation. However, DHICA is efficiently incorporated to the pigmen t, as judged by the high content of carboxylated indole units in natur al melanins. Therefore, the fate of this melanogenic intermediate and the mechanisms of its incorporation to the melanin polymer are major i ssues in the study of melanogenesis. We have recently shown that mouse melanosomes contain two electrophoretically distinguishable tyrosinas e isoenzymes, LEMT and HEMT, that can be purified and completely resol ved (Jimenez-Cervantes et al., 1993a). Herein, we have compared the ab ility of these tyrosinases to catalyze DHICA oxidation. Although highl y purified LEMT shows a very low specific activity for dopa oxidation in comparison to HEMT, it is able to catalyze DHICA oxidation. However the DHICA oxidase activity of HEMT was very low, if significant. The ability of purified LEMT to catalyze DHICA oxidation was abolished by heat, trypsin, or phenylthiourea treatments. LEMT acting on DHICA caus ed the formation of a brownish soluble color similar to DHICA-melanin. Immunoprecipitation of the DHICA oxidase activity of LEMT by specific antibodies suggests that this activity corresponds to TRP1. These res ults indicate that LEMT, most probably identical to the product of the b locus, is a tyrosinase having a specific DHICA oxidase activity. Op posite to HEMT, the true tyrosinase encoded by the albino locus, its r ole in melanogenesis would be related to the incorporation of DHICA in to eumelanin rather than to the first steps of the pathway.