Transfer of tyrosinase to melanosomes in Harding-Passey mouse melanoma

The transfer of tyrosinase from microsomes into melanosomes, without passing through the cytosol in the Harding-Passey mouse melanoma cell, was confirmed by experiments carried out using a combination of radioisotope tracer techniques and immunoprecipitation. 3H-Labeled amino acid incorporation into tyrosinase present in the microsome, melanosome, and soluble fractions confirmed the precursor-product relationship of the enzyme in the microsome fraction and in the melanosome fraction. However, two forms of the enzyme, Ts1- and Ts2-tyrosinase, separated from the soluble fraction by polyacrylamide gel electrophoresis, were shown to play no role in the transfer since little or no incorporation of radioactivity into tyrosinase in this fraction was found. It is suggested that most tyrosinase observed in the soluble fraction does not leak from the melanosomes or the microsomes during homogenization, but comes from necrotic tumor cells. It appears that melanosomal and microsomal tyrosinase might be released from the membrane of necrotic cells modified by various degradation enzymes, considering the data on the recovery of tyrosinase from the soluble fraction, where one-third of total enzyme activity in the postnuclear fraction could not be increased, even when the postnuclear fraction of the tumor was further homogenized radically.     

Photochemical oxidation of tyrosinase

Dye sensitized photo-oxidation inactivates tyrosinases isolated from Neurospora and Agaricus. The rate of inactivation is enhanced by cyanide and is dependent on pH.     

Human tyrosinase is able to oxidize both enantiomers of rhododendrol

Racemic RS‐4‐(4‐hydroxyphenyl)‐2‐butanol (rhododendrol, RD) was used as a topical skin‐whitening agent until it was recently reported to induce leukoderma. We then showed that oxidation of RD with mushroom tyrosinase rapidly produces RD‐quinone, which is quickly converted to RD‐cyclic quinone and RD‐hydroxy‐p‐quinone. In this study, we examined whether either or both of the enantiomers of RD can be oxidized by human tyrosinase. Using a chiral HPLC column, racemic RD was resolved optically to R(?)‐RD and S(+)‐RD enantiomers. In the presence of a catalytic amount of l ‐dopa, human tyrosinase, which can oxidize l ‐tyrosine but not d ‐tyrosine, was found to oxidize both R(?)‐ and S(+)‐RD to give RD‐catechol and its oxidation products. S(+)‐RD was more effectively oxidized than l ‐tyrosine, while R(?)‐RD was less effective. These results support the notion that the melanocyte toxicity of RD depends on its tyrosinase‐catalyzed conversion to toxic quinones and the concomitant production of reactive oxygen species.     

Cobalt tyrosinase: replacement of the binuclear copper of Neurospora tyrosinase by cobalt

The antiferromagnetically spin-coupled copper(II) pair in Neurospora tyrosinase was substituted by cobalt, yielding a stoichiometry of 2 mol of Co/mol of protein. The low magnitude of the high-spin Co(II) EPR signal indicates spin coupling of the two Co(II) ions similar to that observed in the native enzyme. The absorption spectrum with four transitions in the visible region of intermediate intensity (epsilon 607(670), epsilon 564(630), epsilon 526(465)), a shoulder at 635 nm, and the near-infrared bands at 1180 (epsilon 30) and 960 nm (epsilon 15) indicate tetrahedral coordination around the Co(II) center. The cobalt(II) tyrosinase is enzymatically inactive, and there is no evidence that it binds molecular oxygen. Upon addition of cyanide or the competitive tyrosinase inhibitors L-mimosine, benzoic acid, or benzhydroxamic acid te absorption spectrum changes in a characteristic manner. This optical perturbation shows that binding of these inhibitors (and presumably of the substrates) occurs at or near the metal site. One Co(II) ion can be removed preferentially by incubation with KCN at high pH, indicating the two ions not to be in an identical environment.     

Quaternary structure of mushroom tyrosinase

The quaternary structure of $\text{Agaricus}$$\text{bispora}$ tyrosinase has been investigated by sodium dodecylsulfate-acrylamide gel electrophoresis. The enzyme was found to contain two types of polypeptide chains, referred to as Heavy, molecular weight 43,000 ± 1,000, and Light, molecular weight 13,400 ± 600. In aqueous solution the predominant form of tyrosinase m.w. 120,000, has the quaternary structure L₂H₂.     

Hydrodynamic properties of mushroom tyrosinase

The hydrodynamic properties of mushroom tyrosinase were determined at pH 6.5 using a Sephadex G-200 column. From the comparison of its gel-filtration behaviour with those of standard proteins, the following parameters were calculated: MW (122 500 ± 1%), Stokes' radius (42.75 × 10^?8 cm²/sec), diffusion coefficient (5.048 × 10^?7 cm²/sec) and frictional ratio (1.26). These values suggest a globular conformation of this enzyme.