Seasonal variations of Rana esculenta L. skin tyrosinase

Various enzymes are known to be involved in melanin biosynthesis, but the key role appertains to tyrosinase. In amphibians this enzyme displays peculiar characteristics: i) it requires an activation process; ii) its level of enzymatic activity in the animal skin changes depending on the season. In this work, by using chymotrypsin, subtilisin and SDS as putative activators, we studied the activation process of the skin pro-tyrosinase of Rana esculenta L. in different seasons over a period of two years. We found that chymotrypsin and subtilisin were able to yield an active enzyme, but not SDS. The maximum levels of tyrosinase activity were recorded in winter and the minimum in summer. We detected tyrosinase activity in the melanosomal fraction, where the enzyme form was least sensitive to proteolytic activation, probably corresponding to a "mature" tyrosinase. The enzyme forms found in the microsomal and soluble fractions were more sensitive to proteolytic activation, probably corresponding to "immature" tyrosinase. On SDS-PAGE, the tyrosinase activity assays showed a dopa-positive band at 200 kDa and a second aggregated band with a still higher molecular mass. The significance of these results in frog melanogenesis regulation is discussed.