The DHICA oxidase activity of the melanosomal tyrosinases LEMT and HEMT.
Although melanins can be formed in vitro by the unique action of tyrosinase on L-tyrosine, it is now well accepted that other enzymes termed 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-dopachrome yields DHICA, a stable dihydroxyindole with a low rate of spontaneous oxidation. However, DHICA is efficiently incorporated to the pigment, as judged by the high content of carboxylated indole units in natural melanins. Therefore, the fate of this melanogenic intermediate and the mechanisms of its incorporation to the melanin polymer are major issues in the study of melanogenesis. We have recently shown that mouse melanosomes contain two electrophoretically distinguishable tyrosinase isoenzymes, LEMT and HEMT, that can be purified and completely resolved (Jiménez-Cervantes et al., 1993a). Herein, we have compared the ability of these tyrosinases to catalyze DHICA oxidation. Although highly 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 caused 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 results indicate that LEMT, most probably identical to the product of the b locus, is a tyrosinase having a specific DHICA oxidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Jiménez-Cervantes
C
C
Department of Biochemistry and Molecular Biology, School of Medicine, University of Murcia, Spain.
Solano
F
F
Lozano
J A
JA
García-Borrón
J C
JC
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D007211:Indoles / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D007980:Levodopa / Q000378:metabolism; D008544:Melanocytes / Q000201:enzymology*; D008546:Melanoma, Experimental / Q000201:enzymology; D051379:Mice; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D007211:Indoles; D007527:Isoenzymes; D014443:Tyrosine; D007980:Levodopa; C030692:5,6-dihydroxy-2-indolylcarboxylic acid; D014442:Monophenol Monooxygenase