Purification and properties of N‐acetylglucosaminide α1→3‐fucosyltransferase from embryonal carcinoma cells

Abstract

A membrane‐bound α‐l‐fucosyltransferase, which is involved in the synthesis of a developmentally regulated carbohydrate antigen, SSEA‐1, was purified about 2000‐fold from F9 embryonal carcinoma cells. The procedures used were solubilization with Triton X‐100, column chromatography on SP‐Sephadex, DEAE‐Sephadex, RCA‐agarose and on GDP‐agarose. Upon sodium dodecyl sulfate gel electrophoresis, the purified preparation gave a protein band with a relative molecular mass of 65000. The optimum pH of the enzyme was between 6.0 and 7.0 and the Km toward N‐acetyllactosamine was 0.55 mM. The enzyme was active with asialofetuin, but not with intact fetuin. Susceptibility of the product to α‐l‐fucosidase I from almond emulsin verified that the enzyme transferred fucose to C‐3 hydroxyl of N‐acetylglucosamine in the N‐acetyllactosamine structure. Activities of β‐galactoside α1→2‐fucosyltransferase and N‐acetylglucosaminide α1→4‐fucosyltransferase acting on synthetic substrates were not detected in the purified enzyme nor in the crude extract of F9 cells. PYS‐2 parietal endoderm cells lacked all the fucosyltransferases mentioned above. Copyright © 1986, Wiley Blackwell. All rights reserved