The tyrosinase activity of melanosomes from the harding-passey melanoma: The absence of a peroxidase component in vitro

Melanosomes were isolated from the Harding-Passey melanoma with a density gradient technique. Using the Pomerantz radioassay for tyrosinase activity it was found that these isolated melanosomes could hydroxylate tyrosine in the presence of catalase sufficient to deny the enzyme any hydrogen peroxide. It was further found that the rate of hydroxylation was unaffected by the presence of exogenous hydrogen peroxide. Tyrosinase activity could be suppressed by preincubation in diethyldithiocarbamate followed by removal of this inhibitor before enzyme assay. Attempts to regain enzymatic activity, however, by addition of copper II ions were unsuccessful. No peroxidase activity could be detected on the isolated granules, and indeed evidence for a peroxidase inhibitor on the granules was found. It was also found that the peroxidase activity present in a 20% homogenate of mouse muscle did not demonstrate any tyrosinase activity with the Pomerantz assay even in the presence of hydrogen peroxide. It is concluded from these studies that there is tyrosinase on these melanosomes which is capable in vitro of hydroxylating tyrosine without any contribution from an active peroxidase.