Radical production during tyrosinase reaction, dopa-melanin formation, and photoirradiation of dopa-melanin.

It has been suggested that superoxide anion (O2-) may be produced during eumelanin formation and during the photoirradiation of eumelanin , but no direct evidence for this has yet been reported (although O2- production during photoirradiation of pheomelanin has been shown). In this report, the production of O2- was investigated during the formation and photoirradiation of dopa-melanin, a synthetic eumelanin . It was found that cytochrome c was reduced during the tyrosinase reaction and dopa-melanin formation in vitro; this reduction could not be inhibited by superoxide dismutase (SOD). When dopa-melanin was irradiated by UV radiation or by visible light, high nitroblue tetrazolium (NBT) reduction was observed; this reduction was proportional to the light energy and the amount of dopa-melanin. NBT reduction by visible light could be slightly inhibited by SOD, but a 12% decrease of NBT reduction by UV radiation could be shown with the addition of SOD. These observations indicate that some radicals were produced during the tyrosinase reaction and dopa-melanin formation. Further, when dopa-melanin was irradiated, radicals were also produced, some of which were thought to consist of O2-, but others were unknown.

Inhibition of aminoacyl-transfer RNA formation by low-molecular substances from melanoma extract.

Two partially purified fractions of the ethanol precipitate (70-95%) of the water extract of Harding-Passey mouse melanoma, which inhibit protein and DNA syntheses of B-16 melanoma cells in culture, also inhibit protein synthesis in various cell-free systems. By examining their inhibitory effects on limited reactions of protein synthesis, it was found that one of them (ME II) inhibits protein synthesis by blocking aminoacyl-tRNA formation, while the other (ME IV) does not. This inhibition of aminoacyl-tRNA formation was not limited to specific amino acids. Since the amino acid-dependent pyrophosphate (PPi)-ATP exchange reaction catalyzed by aminoacyl-tRNA synthetases was not inhibited, it was concluded that some factor(s) in ME II inhibits amino acid transfer from aminoacyl-AMP to tRNA. ME II contains more than 20 proteins from 10,000 to 90,000 daltons. EDTA treatment of this fraction caused the release of low-molecular substances with inhibitory activity from the proteins. The molecular weights of the active substances are less than 5,000 daltons. The active low-molecular substances are apparently not peptides or nucleotides.

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.

Inhibition of cell-free protein and RNA syntheses by partially purified fractions of mouse melanoma extract.

Ethanol precipitate (70-95%) of the water extract of mouse melanoma (ME) contains suppressors for melanocyte cell division. A fraction which had preferential melanocyte cell line suppressor activity (ME IV2) was shown to inhibit protein synthesis by cell-free systems and RNA synthesis by isolated nuclei of rat liver. The separation and some characterization of the inhibitory factors in ME IV2 were carried out. Upon being boiled, the factor in ME IV2 inhibiting cell-free protein synthesis became inactive, whereas that inhibiting cell-free RNA synthesis remained active. Bio-Gel p-2 column chromatography of ME IV2 gave three distinct fractions (ME IV2-A, -B and -C). ME IV2-A was inhibitory to cell-free protein synthesis but non-inhibitory to cell-free RNA synthesis. On the contrary, ME IV2-B was non-inhibitory to cell-free protein synthesis but inhibitory to cell-free RNA synthesis. ME IV2-C was non-inhibitory to cell-free protein synthesis and seemed to be somewhat inhibitory to cell-free RNA synthesis. Preliminary analyses of the components in these subfractions are also reported.

Inactivation of tyrosinase by dopa.

Tyrosinase in a melanosome is known to be inactivated during melanin formation in vivo, and a similar inactivation was observed in vitro when melanosomes isolated from Harding Passey mouse melanoma were incubated with dopa. Tyrosinase, whether particle bound or in soluble form, was inactivated during the dopa-tyrosinase reaction and the reduction rate of its activity was proportional to the reaction time. Tyrosinase inactivation also occurred when ascorbic acid was added to the reaction system; in which dopaquinone, an oxidation product of dopa which is immediately converted back to dopa by ascorbic acid thus preventing melanin formation. When 14C-dopa or 14C-ascorbic acid were added to the reaction mixture, these radioactive substances were not recovered from the inactivated enzyme protein fraction after incubation. In addition this inactivation of tyrosinase by dopa was not inhibited by any of: 1.4-diazabicyclo[2.2.2]octane, scavenger for singlet oxygen; D-mannitol, that for hydroxyl radical; superoxide dismutase, that for superoxide anion; and catalase, cleavaging enzyme for hydrogen peroxide. Thus the inactivation of tyrosinase appears to be due to neither these radicals, nor reaction products from dopa or ascorbic acid, but to changes in the enzyme itself.