Several genes critical to the enzymatic regulation of melanin production in mammals have recently been cloned and mapped to the albino, brown and slaty loci in mice. All three genes encode proteins with similar structures and features, but with distinct catalytic capacities; the functions of two of those gene products have previously been identified. The albino locus encodes tyrosinase, an enzyme with three distinct melanogenic functions, while the slaty locus encodes tyrosinase‐related protein 2 (TRP2), an enzyme with a single specific, but distinct, function as DOPAchrome tautomerase. Although the brown locus, encoding TRP1, was actually the first member of the tyrosinase gene family to be cloned, its catalytic function (which results in the production of black rather than brown melanin) has been in general dispute. In this study we have used two different techniques (expression of TRP1 in transfected fibroblasts and immunoaffinity purification of TRP1 from melanocytes) to examine the enzymatic function(s) of TRP1. The data demonstrate that the specific melanogenic function of TRP1 is the oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA) to a carboxylated indole‐quinone at a down‐stream point in the melanin biosynthetic pathway. This enzyme activity appears to be essential to the further metabolism of DHICA to a high molecular weight pigmented biopolymer.

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase (EC 1.14.18.1) have been developed. The tyrosine hydroxylase assay uses ~-[carboxy-'~C]tyrosine as the substrate. I4CO2 is released from the products of the hydroxylation and further metabolism of ~-[carboxy-'~C]tyrosine by incubation with ferricyanide, and measured radiometrically. D-Dopa is a preferable cofactor to L-dopa for the assay. Dopa oxidase activity is measured spectrophotometrically. Dopaquinone, produced on the oxidation of L-dopa, reacts with Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone) to form a pink pigment with an absorbance maximum at 505 nm. Details of the optimisation of conditions for the assays and their specificities for the two enzyme activities are described.It is well established that tyrosinase from lower organisms catalyses the first two steps of melanin synthesis: the hydroxylation of tyrosine to dopa, and the oxidation of dopa to dopaquinone [l]. The classical view is that this is also true for the mammalian enzyme (21; it has been suggested that mammalian tyrosinase may catalyse a third step in melanin synthesis: the oxidation of 5,6-dihydroxyindole [3, 41. There have, however, been reports of the separation of melanogenic tyrosine hydroxylase and dopa oxidase activities on purification of the mammalian enzyme [5, 61. To determine conclusively whether the tyrosine hydroxylase and dopa oxidase activities of tyrosinase are inherent in one protein molecule, satisfactory assays for these two activities are required. There are many problems associated with the existing assays so the aim of this work was to develop improved assays for both activities. Tyrosine hydroxylase assayThe tyrosine hydroxylase activity of tyrosinase requires Ldopa, the product of the reaction, as a cofactor. In the absence of cofactor, no activity [7] or very low activity [8] has been reported. In general, a cofactor is required at a low concentration relative to the substrate, but a relatively high concentration of L-dopa is required [9]. If tyrosinase does catalyse the first two steps of melanin synthesis, addition of L-dopa as a cofactor also represents addition of a competitive substrate. Oxidation of L-dopa by dopa oxidase activity will deplete the concentration of cofactor. The requirement for L-dopa as a cofactor thus makes the tyrosine hydroxylase activity of tyrosinase difficult to assay.The Pomerantz method [lo] is used most frequently to assay the tyrosine hydroxylase activity of tyrosinase. It uses ~-[3,5-~H]tyrosine as the substrate and measures the 3 H 2 0Correspondence to A.

Tyrosinase-related protein 1 (TRP1) maps to the brown locus in mice. Although the specific function of TRP1 has been in dispute, mutations in its structural gene result in the formation of brown rather than black melanin. We have investigated the melanogenic function of TRP1 by using immune-affinity purification of the protein and also by using transfection of its gene into fibroblasts to study its characteristics. We show that TRP1 has the ability to oxidize DHICA, a melanogenic intermediate derived from DOPAchrome. In addition, TRP1 has the ability to interact with tyrosinase and significantly stabilize the latter's catalytic function.

No abstract