Investigations on tyrosinase and a tyrosinase related protein

Tyrosinase and tyrosinase-related protein-1 are enzymes involved in melanin synthesis and specific to the melanocyte and the melanoma cell. In the studies upon which this thesis is based, these enzymes were investigated in normal melanocytes, cultured human melanoma cells and transfected mouse fibroblasts. Tyrosinase was isolated from cultured melanoma cells and from bovine eye preparations, and the respective N-terminal amino acid sequences determined. The signal peptide for tyrosinase was defined by comparison of the N-terminal amino acid sequence determined by us and the nucleotide sequence for the tyrosinase gene. Exposure of melanoma cells to enzymes generating active oxygen species (oxygen radicals and hydrogen peroxide), resulted in a dose-dependent increase in tyrosinase activity, an increase blocked by catalase but not by superoxide dismutase or desferrioxamine. Exposure of the cells to aminotriazole, a catalase inhibitor, also resulted in an increase in tyrosinase activity. In studies of the oxygenation of tyrosine by tyrosinase and by oxygen radicals, we found tyrosinase to have no intrinsic peroxidative function, and its oxygenase function to be exclusively specific to the L-isomer of tyrosine. Levels of tyrosinase activity in sera from melanoma patients were determined with a sensitive and stereospecific HPLC procedure, and an RIA was developed for measuring concentrations of tyrosinase protein. Tyrosinase, dopachrome tautomerase and melanin formation were investigated in transfected mouse fibroblasts; those transfected with the albino (c) locus gene expressed an enzyme with tyrosinase acivity, produced melanin precursors, and synthesized phaeomelanin, whereas those transfected with the brown (b) locus gene expressed an enzyme with dopachrome tautomerase activity.