The regulation of the final steps of the melanogenesis pathway, after ~-2-carboxy-2,3-dihydroindole-5,h-quinonc (dopachrome) formation, is studied. It is shown that both tyrosinase and dopachrome tautomerase are involved in the process. In vivo, it seems that tyrosinase is involved in the regulation of the amount of melanin formed, whereas dopachrome tautomerase is mainly involved in the size, structure and composition of melanin, by regulating to the incorporation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into the polymer. Moreover, using ~-3,4-dihydroxyphenylalanine (dopa) and related compounds, it was shown that the presence of dopachrome tautomerase mediates an initial acceleration of melanogenesis since L-dopachrome is rapidly transformed to DHICA, but that melanin formation is inhibited because of the stability of this carboxylated indole compared to 5,6-dihydroxyindole (DHI), its decarboxylated counterpart obtained by spontaneous decarboxylation of L-dopachrome. Using L-dopa methyl ester as a precursor of melanogenesis, it is shown that this carboxylated indole does not polymerize in the absence of DHI, even in the presence of tyrosinase. However, it is incorporated into the polymer in the presence of both tyrosinase and DHI. Thus, this study suggests that DHI is essential for melanin formation, and the rate of polymerization depends on the ratio between DHICA and DHI in the medium. In the melanosome, this ratio should be regulated by the ratio between the activities of dopachrome tautomerase and tyrosinase.In mammals, melanin pigmentation results from the biosynthetic and secretory activity of specialized cells called melanocytes. The main biological function of melanin is in protection against ultraviolet radiation [l], but another possible role for this pigment is to act as a scavenger of cytotoxic agents, such as amines, free radicals and metal ions, preventing undesirable cellular processes [2, 31.The early key steps in the melanogenic pathway, the 0-hydroxylation of c-Tyr to ~-3,4-dihydroxyphenylalanine (dopa) and the oxidation of L-Dopa to L-dopaquinone, are catalyzed by tyrosinase [4, 51. Until 1980, it was believed that the reactions subsequent L-dopaquinone in the pathway leading to melanin, the Raper-Mason pathway, were spontaneous and proceeded through a series of unstable intermediates, such as (2-carboxy-2,3-dihydroindole-5,6-quinone) (Ldopachrome), 5,6-dihydroxyindole (DHI) and 5,6-indolequinone (IndQu) [4-61. However. the content of carboxylic Correspondence to F. Solano,

No abstract

The reduction of 2,3,5-triphenyltetrazolium chloride (TTC) by grapevine cells cultured in suspension was studied in order to assess the reliability of using TTC reduction as a measure of cell viability. Similar to the reduction observed in animals cells, TTC can be reduced in grapevine cells by the cytochrome respiratory path of the mitochondria, although it is mostly reduced (about 72 %) by the alternative respiratory path sensitive to salicylhydroxamic acid. Engagement of the alternative path in TTC reduction was calculated through the √Valt plot, and was established to be 89 %.

Although tyrosinase has been considered for a long time the only enzyme involved in mammalian melanosynthesis, it has been shown that mouse melanoma melanosomes contain high levels of dopachrome tautomerase (DCT2), an enzyme catalyzing DC tautomerization to DHICA. At least in B16 mouse melanoma, DCT is present in higher catalytic amounts than tyrosinase. Moreover, it can be anticipated that tyrosinase and DCT should be very difficult to resolve by most conventional biochemical techniques because of the structural similarity between these enzymes, as predicted from the sequence of their corresponding cDNAs. It is shown that the presence of DCT can cause serious artifacts when tyrosinase activity is determined by most of the currently available methods, such as the Dopa oxidase and melanin formation assays. We describe a simple and convenient method for the preparation of tyrosinase devoid of DCT. The method takes advantage of the different thermal stability of both enzymes. Heating of crude melanosomal extracts at 60 degrees C for 1 hr results in a complete denaturation of DCT, while tyrosinase activity is recovered almost quantitatively. The resulting tyrosinase preparation is considerably purified and the electrophoretic, immunologic and kinetic characteristics of the enzyme appear unaltered. Because if its high yield and simplicity, the method can be used for the microscale partial purification of DCT-free tyrosinase from mammalian malignant melanocytes grown in culture.