The method of formation of black pigment in cells of the nucleus substantiae nigrae has been investigated by employing histochemical techniques which detect tyrosinase, the enzyme responsible for melanin formation in skin melanocytes. Frozen sections of the mid-brains of adult monkeys and cats were incubated in L-tyrosine, to detect the monophenolase activity of tyrosinase; other sections were incubated in L-dopa to detect the diphenolase (dopa oxidase) activity of the enzyme. The dopa, but not the tyrosine, was converted into melanin by the cells of the nucleus substantiae nigrae, resulting in blackening of the cytoplasm.
The intensity of blackening of nigra cells resulting from incubation in L-dopa depended on physical factors. The optimum pH of the reaction ranged between 6.8 and 7.4; the optimum temperature was 37°C; the concentration of substrate employed and the duration of incubation were additional factors which influenced the intensity of the reaction. The conversion of dopa to melanin by nigra cells was prevented by general enzyme inhibitors and by specific inhibitors of tyrosinase, which confirmed the specificity of the reaction for the diphenolase activity of tyrosinase.
The significance of the presence of diphenolase activity, in the absence of monophenolase activity, in adult nigra cells has been discussed in relation to pigment formation in the brain.