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Dopachrome tautomerase (EC 5.3.2.3) catalyses the tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) within the melanin-formation pathway. We have analysed a series of substrate analogues and related compounds as possible substrates and inhibitors of tautomerization. The enzyme appears to be highly specific since D-dopachrome, alpha-methyldopachrome, dopaminochrome, adrenochrome methyl ether and deoxyadrenochrome are not substrates. Conversely, dopachrome tautomerase catalyses the tautomerization of dopachrome methyl ester, suggesting that a carboxy group, either free or as a methyl ester, is essential for enzyme recognition. No inhibition of dopachrome tautomerization was observed in the presence of either semiquinonic compounds, such as tropolone and L-mimosine, or pyrrole-2-carboxylic acid and unsubstituted indole. However, a number of indole derivatives, including DHICA, the product of dopachrome tautomerization, and the analogues 5-hydroxyindole-2-carboxylic and indole-2-carboxylic acid were able to inhibit the enzyme. Furthermore, indoles with a side chain at position 3 of the ring and containing a carboxylic group at the gamma-position of this chain, such as L-tryptophan or indole-3-propionic acid, are stronger inhibitors of the enzyme. Indole-3-carboxylic acid, indole-3-acetic acid and indole-3-butyric acid are very weak inhibitors, showing that the carboxylic group needs to be located at an optimal distance from the indole ring to mimic the carboxylic group at position 2 on the authentic substrate.

No abstract

Tyrosinase induction in murine malignant melanoeytes by gMSH is well known, but its molecular basis has not been characterized. Treatment of BI6 melanoma cells with theophylline or rrMSH mediates a larger induction of tyrosine hydroxylase than of dopa oxidase activity in total cell extracts, and in the melanosomal and microsomal fractions. No evidence for the modulation of a tyrosinase effector was found. SDS-PAGE and slY~ific activity stain demonstrated two forms of tyro.,i~ase, with different degrees of induction by theophylline. These results agree with the recent proposal that two tyrosinases, encoded by different genes, are present in murine melanoeytes. cells in culture trigger the preferential expression of a tyrosinase isoenzyme different from the majority form found in untreated cells. Both forms differ in their electrophoretie mobility in non-reducing SDS-PAGE and in their catalytic properties. The isoenzyme preferentially induced by melanogenic agents displays a larger ratio of tyrosine hydroxylase to dopa oxidase activity, and would therefore mainly affect the first, rate limiting, step of the pathway. This finding conveniently accounts for most of the apparently controversial data in the literature. MATERIALS AND METHODS 2,1. Cell culture attd treatment with melanogenic effectorsBI6-F10 mouse melanoma cells were originally a kind gift of Dr. V. Hearing and have been maintained as described [8], BI6 melanocytes were culttared either on 96-well plates or in 75 cm: flasks from Nunc (Denmark). When using 9f-well plates, cells were seeded at a density of 5,000 ceils per well in 200/.tl of medium. After 24 h, serial dilutions of the melanogenie effectors were added in $0/,tl of medium, and the plate was further incubated for 48 h. Each concentration was assayed in six independent wells the contents of which were pooled by pairs before measurements so as to assay enzymatic activities in triplicate. To obtain higher amounts of cells for subcgllular fraetionation experiments. 5 • I0 S cells were sceclcd in 75 am" flasks, incubated for 24 h and then a volume of medium containing the desired con~ntra-lion of the melanogenic agent was added. Subcellular fractionattonCells were harvested by trypsin treatment and washed twice by centrifugation in homogenization buffer (HB, 10 mM phosphate pH 6.8, 0.25 M sucrose, 0.1 mM EDTA, 0.1 mM PMSF). The washed cells were suspended in HB at about I07 c.ll~'ml and disrupt,-d M a loosefitting, manual, glass-teflon homogenizer, at 4"C. A post-nuclear supernatant was obtained by centrifugation at 700 x g, and further 114Published by Elsevier Science Publishers B, K

The purification of two isoenzymes of tyrosinase has been carried out in Harding-Passey mouse melanoma. One is found in the cytosol and the other one bound to melanosomes. Both migrate as single bands on sodium dodecyl sulphate/polyacrylamide gels, having an apparent Mr of 58 000. Solubilized particulate tyrosinase showed an aggregation equilibrium involving a monomer, tetramer, octamer and a high-Mr micellar form with Brij 35, the solubilizing agent. H.p.l.c. studies indicated a interconversion between those species, the monomer contribution increasing with the sample dilution. The tetramer and the octamer probably represent the predominant forms in vivo. Soluble tyrosinase showed a simpler aggregation equilibrium, involving two forms, monomer and tetramer, with the same interconversion pattern. Fluorescence studies suggested that tryptophan residues were exposed to the aqueous environment when tyrosinase was dissociated by dilution. Tyrosinase shows a tendency to aggregate, at low protein concentration, and a resistance to dissociation by urea or SDS so remarkable that gel-permeation chromatography in 4M-urea does not affect the equilibrium, and the band obtained on SDS/polyacrylamide-gel electrophoresis is a dimer.