CHARACTERIZATION OF STRUCTURAL-PROPERTIES FOR MORPHOLOGICAL-DIFFERENTIATION OF MELANOSOMES .1. PURIFICATION OF TYROSINASE BY TYROSINE AFFINITY-CHROMATOGRAPHY AND ITS CHARACTERIZATION IN B-16 AND HARDING PASSEY MELANOMAS

[摘要] To characterize how structural properties are related to the morphological differentiation of melanosomes, 2 forms of melanosomes, commonly seen in mammals, were isolated from B16 and Harding Passey (HP) mouse melanomas. From these morphologically different melanosomes, tyrosinase was solubilized by BRIJ-35 and purified by affinity chromatographies substituted with tyrosinase substrates. Tyrosine ethyl ester (TEE) and dopa are effective and specific in retaining tyrosinase as an affinity media. Enzyme retrieval with .apprx. 100% recovery is possible by a substrate, TEE, or a competitive inhibitor, N-acetyl-L-tyrosine. The purified tyrosinase of B16 and HP melanosomes possessed a common antigenic site and revealed little difference in size and Km for dopa utilization. Tyrosinase may be involved in melanosome morphogenesis only through melanization; it is not directly related to the architecture, i.e., lamellar and granular patterns, of the inner matrix.