Glycoprotein Biosynthesis

A Golgi apparatus-rich fraction from rat liver was examined for the ability to mediate steps involved in the biosynthesis of glycoproteins.Five enzymatic activities were studied: the transfer of glucosamine, galactose, glucuronic acid, mannose, and N-acetyhreuraminic acid from their sugar nucleotide precursors to endogenous trichloracetic acid-precipitable proteins.The glucosaminyl transfer was enriched 23-fold in the Golgi membrane fraction, and represented 17% of the total activity of the homogenate.The initial rate of transfer of glucosamine exhibits optima both at pH 6.1 and 7.1.UDPglucuronic acid stimulates the net transfer of glucosamine at pH 7.1, but not at pH 6.1.GDP-mannose and GTP are also stimulatory at pH 7.1.UDP-glucose, UDP-galactose, UTP, UDP, and UMP are inhibitory.The Golgi-associated galactosyl transfer was enriched over 24-fold, and accounted for 13 % of the original activity of the homogenate.This transfer activity is stimulated by GDP-mannose.UDP-glucose, UDP-N-acetylglucosamine, and UMP inhibit the transfer of galactose.The Golgi membrane fraction also transfers glucuronic acid from UDP-glucuronic acid to trichloracetic acid-insoluble material.This transfer is stimulated by UDP-N-acetylglucosamine.It represents 5 % of the activity of the original homogenate, and is enriched approximately S-fold in the Golgi fraction.The Golgi-associated transfer of N-acetylneuraminic acid represents 10% of the activity of the homogenate, and is enriched l&fold.This transfer is stimulated by UDPgalactose, and is inhibited by CMP.Although the transfer of mannose from GDP-mannose to trichloracetic acid-precipitable material is very active in the crude homogenate, little transfer, and rapid destruction of GDP-mannose is observed in the Golgi fraction.Mild alkaline treatment (0.5 N NaOH, 24", 90 min) results