Immunologic Homogeneity and Electrophoretic Heterogeneity of Mouse Melanoma Tyrosinases

Abstract

Tyrosinase was solubilized from the 11,000 × g supernatant fraction microsomal and soluble fractions of Harding-Passey mouse melanoma by trypsin digestion and it was partially purified by Sephadex G-100 gel filteration. In contrast to the melanosome fraction, from which only one active tyrosinase could be solubilized, two active components were isolated from the 11,000 × g supernatant fraction, one major faster-eluted component and one minor slower-eluted component. These correspond to T1 and T2 described by Burnett. Both tyrosinases have similar enzyme characteristics, but differ in their isoelectric points. Antiserum was prepared against tyrosinase T1 from melanosomes. This antiserum cross-reacted with both T1 and T2 tyrosinases of the microsomal and soluble fractions in double diffusion plates and immunoelectrophoresis. The precipitin lines produced with T1 or T2 both have tyrosinase activity, which was demonstrated after incubation with L-dopa. These two enzymes have at least one common antigenic peptide sequence at a position remote from the catalytic center.