Glycoprotein Biosynthesis INCORPORATION OF GLYCOSYL GROUPS INTO ENDOGENOUS ACCEPTORS IN A GOLGI APPARATUS-RICH FRACTION OF LIVER

Abstract 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-acetylneuraminic 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. UDP-glucuronic 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-acetyl-glucosamine. It represents 5% of the activity of the original homogenate, and is enriched approximately 8-fold in the Golgi fraction. The Golgi-associated transfer of N-acetylneuraminic acid represents 10% of the activity of the homogenate, and is enriched 18-fold. This transfer is stimulated by UDP-galactose, 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 in the release of 88 to 92% of the radioactivity of the 14C-glucuronic acid-labeled product to the trichloracetic acid-soluble fraction. On the other hand, only 32 to 35% of the 14C-glucosamine-labeled product is converted into a trichloracetic acid-soluble form by this treatment. When glucosamine transfer is stimulated by the addition of UDP-glucuronic acid, however, approximately 50% of the product is released by alkali. It is suggested that the alkali-stable products might represent glycoproteins, while the more alkali-labile products may be glycosaminoglycans. Bacterial heparinase degraded 70% of the glucuronic acid-labeled product, and 25% of the glucosamine-labeled product. Testicular hyaluronidase degraded 35% of the glucuronic acid-labeled, and 7% of the glucosamine-labeled material. Pronase digestion converted all of the radioactivity of glucosamine-, glucuronic acid-, or galactose-labeled trichloracetic acid-precipitable products into trichloracetic acid-soluble forms. These digestion products are still of relatively high molecular weight, as they are eluted in the void volume of a Sephadex G-50 column. Acid hydrolysis released all of the radioactivity of the glucosamine-labeled, pronase-digested product as free glucosamine, as identified by paper chromatography. The radioactive product of the galactosyl transferase reaction was shown to be 14C-galactose by the same method. The results of this study are consistent with the hypothesis that the N-acetylglucosamine, galactose, and N-acetyl-neuraminic acid residues of the terminal sialic acid → galactose → N-acetylglucosamine sequence of many glycoproteins are added to the nascent glycoprotein within the Golgi apparatus, while the sugar residues of the inner core are inserted at other sites in the endoplasmic reticulum.