BIOCHEMICAL-CHARACTERIZATION OF THE MELANOGENIC SYSTEM IN THE EYE OF ADULT RODENTS

The melanogenic activities in the eye of the adult gerbil (Meriones un guiclatus) have been investigated and compared to those found in the B 16 mouse melanoma model. Eye extracts contain tyrosine hydroxylase, DO PA oxidase, DOPAchrome tautomerase and DHICA oxidase activities. The s ubcellular distribution of these activities was investigated by differ ential centrifugation and detergent solubilization of the particulate fractions. The distribution pattern closely resembled the one found fo r mouse melanoma, with a higher percentage of activity associated to t he particulate fractions but a substantial proportion in the cytosolic fraction. The tyrosine hydroxylase activity was characterized by a K- M of 62 mu M for L-tyrosine and a stringent requirement for the co-fac tor L-DOPA (K-a 10.3 mu M) The K-M for L-DOPA was 0.41 mM. The sensiti vity of the eye and mouse melanoma tyrosinase activity to a variety of substrate analogs and metal chelators was found to be identical. In k eeping with these kinetic similarities, eye tyrosinase displayed some structural properties resembling those of the melanoma enzyme. The mol ecular weight of the enzyme, determined by SDS-PAGE and DOPA oxidase a ctivity stain, was 75 kDa for the eye enzyme and 66.2 kDa for melanoma tyrosinase, and both enzymes were apparently dimeric in non ionic det ergent solution. Immunoprecipitation with specific antibodies proved t hat at least 80% of the total tyrosinase activity could be immunopreci pitated with the specific anti-tyrosinase antibody alpha PEP7, while t he anti-TRP-l monoclonal antibody TMH-1 precipitated little, if any, t yrosinase activity. Taken together, these observations provide the fir st vis-a-vis comparison of an extracutaneous melanogenic system and th e melanogenic system of melanoma. Our results prove that, at least in rodents, the melanogenic system in the eye is similar, but not identic al, to the melanin biosynthesis machinery of epidermal melanocytes.