Action of Trypsin and Detergents on Tyrosinase of Normal and Malignant Melanocytes 1

Summary

The T₁ variety of tyrosinase is present in both particulate and soluble or readily solubilized forms in the pigmented hypodermis (hair bulbs) of C57BL mice and Harding-Passey mouse melanoma. Trypsin treatment of 35,000g supernatants containing the microsomal (small granule) fraction of gentle homogenates of hair bulbs and melanoma results in significantly increased T₁ activity within polyacrylamide gels. Similar treatment of 100,000g supernatants results in a slight increase in T₁ activity. Addition of Triton-X or DOC to 35,000g supernatants of hair bulb and melanoma homogenates followed by centrifugation at 100,000g results in a marked enhancement of T₁ when the latter supernatants are treated with trypsin. In the absence of trypsin treatment, T₁ activity is comparable to nondetergent-treated controls. A slow-moving dopa-reactive band (T_s) is found in electropherograms of the nontrypsinized 100,000g supernatants of detergent-treated 35,000g supernatants. It is absent in those treated with trypsin. The slow-moving enzyme appears to give rise to T₁ molecules when eluted from acrylamide gels and even to a greater extent when elution is combined with trypsin treatment prior to reelectro-phoresis. In mammals, tyrosinase apparently is not derived by a proteolytic activation of protyrosinase.