In cultured melanotic
melanoma, a marked decrease of pigmentation has been found to be induced by the addition of
tunicamycin [Y. Mishima and G. Imokawa (1983) J. Invest. Dermatol. 81, 106-114]. Since it appears that this impaired pigmentation arises from the loss of
asparagine-linked
sugar chains serving as a signal for transport of
tyrosinase from GERL (Golgi-associated endoplasmic reticulum of lysosomes) to premelanosomes, tyrosinases from the membrane fraction of Greene's hamster
melanoma have been purified, and the structures of their
sugar chains have been analyzed. Two kinds of tyrosinases were purified by
Triton X-100 solubilization;
DEAE-cellulose,
Sephadex G-200, and
DEAE-Sephadex column chromatography; and preparative
polyacrylamide gel electrophoresis. The two tyrosinases were separated by
polyacrylamide gel electrophoresis, and both corresponded to Mr 69,000. Their
asparagine-linked
sugar chains were released by hydrazinolysis and analyzed. The
sugar chains of the two tyrosinases were identical except for the
sialic acid contents. One mole of each
tyrosinase contained 1 mol of high-
mannose-type
sugar chains and 3 mol of complex-type
sugar chains. The former chain has Man3 approximately 5 X GlcNAc2 and the latter has Man3 X GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc as their core structures. The complex-type
sugar chains are composed of mono-, bi-, tri-, and tetraantennary
sugar chains, with +/- Sia alpha 2----3Gal beta 1----4GlcNAc beta 1----as their outer chains.