from lower organisms such as mushroom can be purified to homogeneity with low yield and is commercially available. Similarly, mammalian tyrosinase has been purified from various sources such as pigmenting melanoma cells. Purification of the authentic enzyme is, however, complex and results in heterogeneous products with varying degrees of solubility. These purifications are usually not scalable and unlike recombinant produced material, mutations cannot be introduced to study the biochemistry of disease-related proteins. More recently, Kong et al. have reported expression of first 455 amino acid residues of human tyrosinase, the so-called ectodomain, in E. coli. As expected from expression in a prokaryote, this soluble form of tyrosinase is not glycosylated and the purification yield was low. While this recombinant protein exhibited both tyrosine hydroxylation and oxidation TG-101348 activities, its biochemical properties were different from those reported for other mammalian enzymes including a high temperature maximum for activity and a lower specific activity. Our attempts to produce active intra-melanosomal domain, hTyrCtr, in E. coli failed probably due to protein misfolding. Switching to the baculovirus system resulted in the production of soluble expressed protein. The purified hTyrCtr is enzymatically active and has biochemical properties similar to the published data, is 26148857 post-translationally modified, and is optimally active at 37uC and neutral pH. Tyrosinase undergoes highly-regulated and complex processing in the endoplasmic reticulum and Golgi apparatus on the way to its final destination in the melanosome, a specialized endosomal organelle. Bhatnagar et al. found that melanosomes purified from a mouse melanoma cell line had an internal acidic pH of,4.6. Similarly, Puri et al. found that C57BL6 mouse melanosomes were acidic but that the mutation in OCA2, a cause of albinism, resulted 
in isolated vesicles now having an internal neutral pH. Counter to these findings, Ancans et al. reported that melanin synthesis occurred at neutral pH and proposed an alternative explanation for the effect of the OCA2 mutation. These conflicting results may not, however, be mutually exclusive of one another. Our in-vitro data indicates that although the pH optimum of human tyrosinase is 7.4 it is still active below pH 6.0 and, thus, in-vivo would be expected to function under potential sub-optimal conditions in the melanosome. Hence, the pH activity profile and pH optimum of purified enzyme are consistent with in-vivo activity under the physiological extremes reported in the literature. Part of tyrosinase maturation is its post-translational Nglycosylation. Branza-Nichita and colleagues have reported that glycosylation is critical for tyrosinase folding, quality control and, therefore, activity. Similarly, Imokawa et al. found that drugs that inhibit N-linked glycosylation inhibit tyrosinase and melanin production in melanoma cell lines. These data are consistent with our molecular modeling, which shows glycosylated residues are located on the surface of the intra-melanosomal portion of tyrosinase, and so are accessible for interactions with other melanosomal proteins. OCA1B Mutations Alter Tyrosinase Activity Glycosylation may, in fact, serve as a control point in regulating cellular tyrosinase activity. Ujvari et al. found that the rate 2435173 of translation of tyrosinase, in part, determined its level of Nglycosylation. Our purified enzyme is glycosylated at
