The Tyrosinases of Mouse Melanoma ISOLATION AND MOLECULAR PROPERTIES

Abstract An effective technique has been developed for isolating and purifying the tyrosinases of mouse melanoma. The soluble tyrosinases normally present in the crude homogenate of melanoma tissue have been separated from the other proteins of the homogenate by repeated electrophoresis in urea-containing polyacrylamide gel systems. The amount of purified, soluble enzyme recovered by this technique is essentially equal to that initially present in the supernatant of an homogenate of freshly excised or fresh frozen mouse melanoma tissue. Three distinct forms of active tyrosinase (EC 1.10.3.1) were found: two soluble and one insoluble. T11 and T2 were obtained from the supernatant of homogenized tissue and purified by the technique described here. T3, bound to particulate material, was separated from the supernatant of the homogenate; it can be partially solubilized with detergent, as described elsewhere. The final preparations of T1 and T2 were judged to be free from contamination with other proteins because their elution profiles from preparative polyacrylamide gel electrophoresis were symmetrical; the specific activity of their elution fractions was consistently high; the pattern of enzymic bands corresponded to that of protein bands after analytical polyacrylamide gel electrophoresis under at least three different conditions of "running pH" or in gels of varied acrylamide content; and, in equilibrium ultracentrifugation experiments, the plot of the logarithm of the net fringe displacement versus the comparator x-coordinate was a straight line. T1 and T2 were found to have similar molecular weights (66,000 and 56,700, respectively) as determined by equilibrium ultracentrifugation, they differed in amino acid composition, and they did not appear to undergo interconversion. As determined from optical rotatory dispersion and circular dichroism spectra, the apparent α-helical content of T1 and T2 was approximately 28% and the helical configuration was resistant to changes of temperature. Concurrent studies revealed that enzymic activity was much more sensitive than helical configuration to temperature change. When T1 and T2 were treated with dithiothreitol and the excess dithiothreitol was removed, their enzymic activity was reduced or entirely lost; it was partially restored or augmented by placing the enzyme preparation in an environment favorable to oxidation. During reduction and reoxidation, T1 and T2 appeared to retain their characteristic, initial electrophoretic mobility. As determined spectrophotometrically and manometrically, neither form of soluble, purified tyrosinase was activated by chlorpromazine. Small quantities of active tyrosinases with a molecular weight of approximately 30,000 were obtained from melanoma by Sephadex column chromatography. Also, the amino acid content of the enzymes prepared in this way was about half that of the T1 and T2 prepared by preparative polyacrylamide gel electrophoresis. The more abundant and possibly more stable forms, T1 and T2, may, therefore, be dimers. The enzymes prepared by Sephadex column chromatography were judged to be free from contamination both because the specific activity of the fractions of each elution profile was high and did not change as the result of additional physical and chemical treatment, and because the sedimentation pattern showed a single, symmetrical peak.