This research demonstrates that the melanosome proteins extracted from the pigmented macrophages (Kupffer Cells: KC) of R. esculenta L. liver possess a tyrosinehydroxylase (TH) activity. The presence of a TH together with a dopaoxidase (DO) activity, in the melanosome proteins, shows that the melanin pathway of these cells is carried out by a tyrosinase. This tyrosinase possesses peculiar physical-chemical and kinetic characteristics unlike those of skin or melanoma cell tyrosinases. The TH reaction depends on catalytic dopa as cofactor, it is not affected by catalase or H2O2, showing that it is catalysed by the tyrosinase and not by the peroxidase present in melanosomes. Activation experiments have demonstrated that the enzyme is activated by Cu++ ions, and not by activators effective on the other tyrosinases. SDS totally inhibits TH activity even under the critical micellar concentration, suggesting that tyrosinase inhibition mechanism differs from that of the other known tyrosinases. Immuno-gold analysis on the Kupffer Cells shows that tyrosinase is recognized in the trans-Golgi vesicles, and both on the outside and into the premelanosomes and melanosomes. Western-blotting analysis demonstrates that anti-KC-tyrosinase antibodies recognize two principal bands of 260 KD and 121 KD. Activity tests show that tyrosinase activity is limited to the 260 KD band, indicating that the enzyme activity needs to form a molecular aggregate to become manifest. Investigation on the physical structure of the liver melanin shows that the biopolymer may be described as a network of group of nanoclusters having different sizes and based on a few number of units of indolic type.
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|Titolo:||The melanogenic system of the liver pigmented macrophages of Rana esculenta L|
|Data di pubblicazione:||2005|
|Appare nelle tipologie:||4.1 Contributo in Atti di convegno|