Purification of hamster melanoma tyrosinases and structural studies of their asparagine-linked sugar chains

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Abstract

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 Man3~5 · GlcNAc2 and the latter has Man3 · GlcNAcβ1 → 4(±Fucα1 → 6)GlcNAc as their core structures. The complex-type sugar chains are composed of mono-, bi-, tri-, and tetraantennary sugar chains, with ±Siaα2 → 3Galβ1 → 4GlcNAcβ1 → as their outer chains.

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    This study was supported in part by a Grant-in-Aid for Cooperative Research and for Cancer Research of the Ministry of Education, Science, and Culture of Japan. This paper is a part of the dissertation submitted by T.O. to Kobe University School of Medicine for the degree requirement of Doctor of Medical Sciences.

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