Tyrosinase activity in the medium of human melanoma cell cultures.
The medium of cultured melanoma cells was studied for tyrosine hydroxylation and dopa-oxidizing activity. The supernatant obtained after centrifugation at 100 000 g for 2 hours was treated with ammonium sulphate, and the precipitate obtained between 35 and 50% saturation was used. Dopa was determined as the product of tyrosine hydroxylation and 5-S-cysteinyldopa as the product of dopa oxidase activity. Determinations were performed with HPLC and electrochemical detection. Our preparation of culture medium of cells showed the following. 1) No hydroxylation of tyrosine in the absence of co-factor. 2) Hydroxylation of L-tyrosine in the presence of dopamine. No hydroxylation with boiled medium. Minimal effect of catalase on hydroxylation. 3) Hydroxylation of tyrosine in the presence of ascorbic acid. Hydroxylation was catalyzed also with boiled medium. Catalase strikingly diminished hydroxylation. 4) Oxidation of L-dopa to dopaquinone determined as its main reaction product with cysteine, 5-S-cysteinyl-dopa. There was negligible oxidation with boiled medium. 5) With dopamine as co-factor the catalysis of tyrosine hydroxylation was stereospecific for L-tyrosine. Dopa oxidase activity was also stereospecific for L-dopa.
D001205:Ascorbic Acid / Q000494:pharmacology; D004156:Catechol Oxidase / Q000032:analysis*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media / Q000032:analysis; D004295:Dihydroxyphenylalanine / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D008545:Melanoma / Q000032:analysis*; D014442:Monophenol Monooxygenase / Q000032:analysis*; D010084:Oxidation-Reduction; D013237:Stereoisomerism; D014443:Tyrosine / Q000378:metabolism
D003470:Culture Media; D014443:Tyrosine; D004295:Dihydroxyphenylalanine; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase; D001205:Ascorbic Acid