Purification and properties of N-acetylglucosaminide alpha 1----3-fucosyltransferase from embryonal carcinoma cells.
A membrane-bound alpha-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 65 000. 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 alpha-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 beta-galactoside alpha 1----2-fucosyltransferase and N-acetylglucosaminide alpha 1----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.
H
H
Muramatsu
Y
Y
Kamada
T
T
Muramatsu
D000606:Amino Sugars / Q000378:metabolism; D000818:Animals; D001212:Asialoglycoproteins; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D060748:Fetuins; D005647:Fucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D006602:Hexosyltransferases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D009373:Neoplasms, Germ Cell and Embryonal / Q000201:enzymology*; D000509:alpha-Fetoproteins / Q000378:metabolism; D005644:alpha-L-Fucosidase / Q000378:metabolism
D000606:Amino Sugars; D001212:Asialoglycoproteins; D060748:Fetuins; D000509:alpha-Fetoproteins; C017029:asialofetuin; C000458:N-acetyllactosamine; D008715:Methionine; D005647:Fucosyltransferases; D006602:Hexosyltransferases; C031692:galactoside 3-fucosyltransferase; C020559:Glycoprotein 6-alpha-L-fucosyltransferase; D005644:alpha-L-Fucosidase; D005033:Ethylmaleimide