Biochemical characterization of the melanogenic system in the eye of adult rodents.

The melanogenic activities in the eye of the adult gerbil (Meriones unguiculatus) have been investigated and compared to those found in the B16 mouse melanoma model. Eye extracts contain tyrosine hydroxylase, DOPA oxidase, DOPAchrome tautomerase and DHICA oxidase activities. The subcellular distribution of these activities was investigated by differential centrifugation and detergent solubilization of the particulate fractions. The distribution pattern closely resembled the one found for mouse melanoma, with a higher percentage of activity associated to the particulate fractions but a substantial proportion in the cytosolic fraction. The tyrosine hydroxylase activity was characterized by a KM of 62 microM for L-tyrosine and a stringent requirement for the co-factor L-DOPA (Ka 10.3 microM). The KM for L-DOPA was 0.41 mM. The sensitivity of the eye and mouse melanoma tyrosinase activity to a variety of substrate analogs and metal chelators was found to be identical. In keeping with these kinetic similarities, eye tyrosinase displayed some structural properties resembling those of the melanoma enzyme. The molecular weight of the enzyme, determined by SDS-PAGE and DOPA oxidase activity stain, was 75 kDa for the eye enzyme and 66.2 kDa for melanoma tyrosinase, and both enzymes were apparently dimeric in non ionic detergent solution. Immunoprecipitation with specific antibodies proved that at least 80% of the total tyrosinase activity could be immunoprecipitated with the specific anti-tyrosinase antibody alpha PEP7, while the anti-TRP-1 monoclonal antibody TMH-1 precipitated little, if any, tyrosinase activity. Taken together, these observations provide the first vis-à-vis comparison of an extracutaneous melanogenic system and the melanogenic system of melanoma. Our results prove that, at least in rodents, the melanogenic system in the eye is similar, but not identical, to the melanin biosynthesis machinery of epidermal melanocytes.

Melanin formation in the inner ear is catalyzed by a new tyrosine hydroxylase kinetically and structurally different from tyrosinase.

Detergent solubilized extracts of the cochleae of adult gerbils (Meriones unguiculatus) contain a tyrosine hydroxylase activity measurable by the radiometric method of Pomerantz. This activity is not related to Fenton-type reactions, since it is not inhibited by free radical scavengers and is heat and protease sensitive. It does not appear to be related to a peroxidase (EC 1.11.1.7) since it is neither dependent on H2O2, nor inhibited by catalase (EC 1.11.1.6). The involvement of a tyrosine hydroxylase (EC 1.14.16.2) related to catecholamine synthesis is also unlikely, since the activity is highly sensitive to 2-mercaptoethanol and is not increased by addition of tetrahydrobiopterin. The activity in crude inner ear extracts displayed an unusual maturation behaviour, with a slow activation upon aging at 4 degrees C. Fully active enzyme displayed Michaelis-Menten kinetics, with a Km for L-tyrosine of 47 microM. Cochlear tyrosine hydroxylase, but not melanoma tyrosinase (EC 1.14.18.1), was inhibited by o-phenanthroline, and was not dependent on L-DOPA as cofactor for full enzymatic activity. Crude extracts were also able to catalyze L-DOPA oxidation and melanin formation from either L-tyrosine or L-DOPA. The tyrosine hydroxylase, DOPA oxidase and melanin formation activities most probably resided in the same molecule, as suggested by inhibition studies. A tyrosine hydroxylase and melanin formation activity with identical properties was found in primary cultures of stria vascularis melanocytes. Immunochemical evidence confirmed the absence of either the tyrosinase encoded for by the albino locus, or the tyrosinase isoenzyme TRP1, encoded for by the brown locus. Conversely, an immunorreactive band of molecular weight 70 kDa was specifically recognized by a tyrosinase polyclonal antiserum in Western blot experiments. These results prove that melanogenesis in the cochlea, and likely in other extracutaneous locations such as the brain, is catalyzed by enzymatic systems different from, but related to tyrosinase.