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.