Post-tyrosinase regulators of melanin synthesis ten years after

Solano F, Garcia-Borron J.C., Aroca P, Lozano J~I.
Dpto. Bioquimica y Biologia MolecuEar, Facultad de Medicina
Universidad de Murcia, Spain

The first indication of post-tyrosinase regulators of melanin synthesis came from a study of the inhibition of melanogenesis by melatonin in the hair follicle of siberian hamsters (1). However, it was not until 1980 that the possible nature and activity of such factors was postulated in a classical paper by Dr. John Pawelek's group (2), when three "distal factors" were postulated :

a dopachrome conversion factor (DCF), that was thought to accelerate the conversion of dopachrome to 5,6-dihydroxyindole (DHI);

a 5,6-DHI conversion factor expressed by cells stimulated by MSH and catalyzing the conversion of DHI to melanin;

a 5,6-DHI blocking factor restricting melanogenesis at 5,6-DHI.

After ten years of active investigation by several teams the most important of these three factors appears to be DCF, since a) dopachrome conversion is a critical step in the biosynthesis of melanins, as noted by Dr. Prota and coworkers (3); b) 5,6-DHI conversion activity can be accounted for by tyrosinase (4); and therefore a new factor does not need to be involved to explain the increased rate of DHI evolution in cells stimulated by MSH; c) as far as we know, no clear cut evidence of the existence of a DHI blocking activity has ever been put forward. This activity has been always measured in crude extracts and no partial purification and characterization have been published. Moreover, its mechanism of action appears difficult to postulate on chemical backgrounds and, as first noted by Dr. King's group at Minnesota, its postulated activity might be accounted for by DCF(5). We will therefore restrict our discussion to the recent findings on dopachrome conversion activity.
There is a rather general agreement as to the nature of the reaction catalyzed by dopachrome. The factor accelerates the conversion of dopachrome into the colorless compound 5,6-DHICA, as first suggested by Korner & Pawelek (6) and latter on unambiguously demonstrated by a variety of techniques including nuclear magnetic resonance and mass spectrometry (7), HPLC (8,9), and spectrophotometric and radiometric criteria (10,11). Since the transformation of dopachrome into DHICA is a tautomerization, the name dopachrome tautomerase (E.C.5.3.2.3) appears as the most precise and informative and should be preferred to other possible denominations (11). Although there are no doubts about the fact that the spontaneous evolution at neutral pH of the rather unstable compound dopachrome leads to DHI (12), while the action of dopachrome tautomerase leads to the non-decarboxy]ative rearrangement to DHICA, this very fact has prompted some doubts and controversy as to the actual nature of the catalytic factor. It has been clearly shown that several metal ions, including Cu(ll). Co(II) and mostly Ni(II), catalyzed the non-decarboxylative rearrangement of dopachrome to DHICA (3,8,13), and the contents of some of these ions is high in melanoma tissues and probably in the melanosomes of normal melanocyrrs. Moreover; the total carboxylic group contents of melanins is higher when the pigment is synthesized in the presence of metal ions (14), and these melanins resemble more closely the natural pigment, whose carboxylic group contents is also high (14,15). It has therefore been postulated that the actual regulators of dopachrome rearrangement within the melanocytes could be metal ions, where natural and relative concentrations might account for some of the differences in natural melanin obtained from various sources.

Should we therefore come back to the first idea that tyrosinase is the only enzymatic factor controlling melanosynthesis from tyrosine ? We think that, although the catalytic effect of metal ions on dopachrome rearrangement are indisputable (3,8,13), their actual importance in vive is not clear. On the other hand, the evidence pointing to the existence of a specific protein, dopachrome tautomerase, within the melanocyte is overwhelming, and even be summarized as follows :

- the activity has been partially purified by a variety of chromatographic techniques to high specific activity (5,9,11). Dr. King's group has reported the purification of the enzyme to electrophoretic homogeneity (XIVth International Pigment Cell Conference, Kobe). The finding of active and inactive chromalographic fractions i.e. of a specific chromatographic profile excludes the possibility that the activity might be explained by a contaminatjon of buffers by metal ions;

- the activity is heat-sensitive and protease-sensitive as shown by a variety of authors;

- the activity of purified preparations is not affected by the inclusion in the assay media of high concentrations of EDTA (11) or by Chelex 100 treatment of buffers (8);

- dopachrome tautomerase is competitively inhibited by some analogues of the substrate and product including L-Trp (11) and DHICA itself (16);

- purified dopachrome tautomerase is highly stereospecific and does not catalyze the decarboxylative rearrangement of D-dopachrome (16);

- the hydrolytic action of a variety of glycosidases activates the dopachrome tautomerase activity (results to be published). All these observations leave little if any doubt as to the actual existence of a specific enzyme, specially if one considers that dopachrome tautomerase activity is high, and, in B16 melanoma melanocytes, comparable in terms of units/g of tissue to tyrosinase activity. In light of the available evidence. two matters appear therefore settled : the existence of a distinct protein catalyzing dopachrome conversion, and the nature of its reaction product, DHICA, which immediately sets the name dopachrome tautomerase as the most appropriate for the protein. However, several points await further clarification and might be the subject of future research.

We will just mention three of them.

1. As we mentioned above, both metal ions and dopachrome tautomerase are able to catalyze the same reaction, and the concentration of both types of reagents appear to be high in the melanosomes. It will be therefore important to determine which one of the two is the actual physiological regulator of dopachrome evolution, although their action is not mutually exclusive and might even be synergistic (13).

Research in this field should undoubtly be speeded up by the collaboration between the different teams involved and by the standardization of the assay methods for metal ions and dopachrome tautomerase activity.

2. The relationship between dopachrome tautomerase and indole blocking factor first noted by the Minnesota group (5) is also interesting. In the presence of dopachrome tautomerase (or metal ions) the evolution of dopachrome leads to polymeric products more soluble and less dark than the ones obtained spontaneously (5,11). and similar to DHICA-melanins (15). Visually, dopachrome tautomerase: behaves as an indole blocking factor, although we have shown that the enzyme actuallv increase the rate of formation of a melanin-like polymer, in systems containing elther- tvrosine or dopachrome and tyrosinase and catalytic amounts of dopa (11.13, results to be published). It is therefore possible that the postulated indole blocking activity might be an artifact of visual or spectrophotometric estimates of melanin formation rates, and might be accounted for by the formation of DHICA-melanins, rather than DHI-melanins in the presence of the enzyme. A definitive answer might come from a more systematic search of the blocking activity and its comparison to the tautomerase activity.

3. In addition, a protein factor that shows strong similarities to the specificity of dopachrome tautomerase has been described in insects (17). This factor has been named dopaquinone imine conversion factor. Therefore; it is plausible that enzymes catalyzing the tautomerization of dopachrome could occur in different organisms of the phylogenetic scale. The widespread existence of such enzyme would support the view that it might play an important role in the regulation of melanin biosynthesis.

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