Radioassay for Oxidation of Tyrosine by Tyrosinase using L-[Ring-^3H] tyrosine and L-[Carboxyl-^<14>C] tyrosine

概要

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Two intermediates involved in the synthesis of melanin from L-tyrosine by mushroom tyrosinase (EC 1.14.18.1) were assayed simultaneously by using L-[ring-^3H] tyrosine and L-[carboxyl-^<14>C] tyrosine. Tritium ions released from the 3-position of L-[3,5-^3H] tyrosine or the 6-position of L-[2,6-^3H] tyrosine were isolated as ^3H_2O and employed to determine the quantity of L-dopa or leucodopachrome, respectively (^3H_2O method). 5,6-Dihydroxyindole formed at the decarboxylation step was estimated from the radioactivity remaining after the evolution of ^<14>CO_2 from L-[carboxyl-^<14>C] tyrosine (^<14>CO_2-release method). The lower limits of determination in the ^3H_2O and the ^<14>CO_2-release methods were 0.1 pmol and nmol of the intermediates, respectively. The formations of L-dopa, leucodopachrome, and 0.1 5,6-dihydroxyindole were measured at various concentrations of L-tyrosine by both methods, and dopachrome was determined colorimetrically by measuring the absorbance changes at 475nm. The maximum velocities were estimated from Lineweaver-Burk plots.
社団法人日本薬学会の論文
1982-06-25