Characterization of Structural Properties for Morphological Differentiation of Melanosomes: I. Purification of Tyrosinase by Tyrosine Affinity Chromatography and its Characterization in B16 and Harding Passey Melanomas

To characterize how structural properties are related to the morphological differentiation of melanosomes, two 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. We found that tyrosine ethyl ester (TEE) and dopa are effective and specific in retaining tyrosinase as an affinity media and that enzyme retrieval with approximately 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 the Mikaelis constant for dopa utilization. It is likely that tyrosinase is involved in melanosome morphogenesis only through melanization, and that it is not directly related to the architecture, i.e., lamellar and granular patterns, of the inner matrix.