PREPARATION OF PURIFIED TYROSINASE DEVOID OF DOPACHROME TAUTOMERASE FROM MAMMALIAN MALIGNANT MELANOCYTES

Although tyrosinase has been considered for a long time the only enzym e involved in mammalian melanosynthesis, it has been shown that mouse melanoma melanosomes contain high levels of dopachrome tautomerase (DC T2), an enzyme catalyzing DC tautomerization to DHICA. At least in B16 mouse melanoma, DCT is present in higher catalytic amounts than tyros inase. Moreover, it can be anticipated that tyrosinase and DCT should be very difficult to resolve by most conventional biochemical techniqu es because of the structural similarity between these enzymes, as pred icted from the sequence of their corresponding cDNAs. It is shown that the presence of DCT can cause serious artifacts when tyrosinase activ ity is determined by most of the currently available methods, such as the Dopa oxidase and melanin formation assays. We describe a simple an d convenient method for the preparation of tyrosinase devoid of DCT. T he method takes advantage of the different thermal stability of both e nzymes. Heating of crude melanosomal extracts at 60-degrees-C for 1 hr results in a complete denaturation of DCT, while tyrosinase activity is recovered almost quantitatively. The resulting tyrosinase preparati on is considerably purified and the electrophoretic, immunologic and k inetic characteristics of the enzyme appear unaltered. Because if its high yield and simplicity, the method can be used for the microscale p artial purification of DCT-free tyrosinase from mammalian malignant me lanocytes grown in culture.