The distribution of the three glycosyltransferases synthesizing the terminal trisaccharide sialic acid ---> D-galactose -o N-acetylglucosamine present in many glycoproteins was determined in Golgi fractions prepared from rat liver homogenates by a modification of the procedure of Ehrenreich et al. (1973, J. Cell Biol. 70:671-684) . The enzymes were assayed with asialofetuin, ovomucoid, and Smith-degraded ovomucoid as sugar acceptors . Careful adjustment of the pH of all sucrose solutions to 7 .0 ± 0.1 prevented enzyme inactivation, and allowed quantitative recoveries at every isolation step. The three morphologically and functionally different Golgi fractions GF 1, GF2 , and GF 3 showed (in that order) decreasing specific activities of all three enzymes, but the relative amounts and relative specific activities of the three transferases in any given fraction were nearly identical . Two marginal fractions, one extra heavy (collected on the gradient below GF 3) and the other extra light (isolated by flotation from the postmicrosomal supernate) were found to contain recognizable Golgi elements . An enrichment of any transferase over the two others was not detected in either preparation .A partial release of content from a combined GF1+2 was achieved by treatment with the nonionic detergent Triton X-100. Low Triton/phospholipid ratios (<2 mg/mg) led to lysis of the vesicles and cisternae and loss of very low density lipoprotein particles (ascertained by electron microscopy), but failed to separate the transferases from each other; the three enzymes sedimented together with a population of empty vesicles to a density of -1 .08 g/ml .KEY WORDS rat liver Golgi fractions subfractionation detergents (Triton X-100) sialyltransferase " galactosyltransferase n-acetylglucosaminyltransferaseThe Golgi complex plays a major (perhaps exclusive) role in the terminal glycosylation of glycoproteins (32,46), in addition to its involvement in the intracellular transport, concentration, and partial proteolytic processing of secretory proteins (27,41) . The evidence is firm for exportable glycopro-J . CELT. BIOLOGY

1. Galactosyltransferase activities in postnuclear supernatants and Golgi fractions from rat liver were assayed with two improved and simplified methods, using high-and low-molecular-weight acceptors. Transfer to N-acetylglucosamine was measured after the separation of the reaction product N-acetyllactosamine from all other radioactive molecules (including galactose) on an ionexchange column partially converted to the borate form. To determine the transfer of galactose to a glycoprotein acceptor we used ovomucoid, which accepts galactose without any previous chemical or enzymic modification.2. Both enzymic activities were enriched 60 -80-fold (compared with the post-nuclear supernatant) in Golgi fractions, which were isolated on two subsequent sucrose gradients and identified morphologically by their high contents of stacked Golgi elements. The two activities could not be resolved by isolation of the Golgi fractions or by detergent solubilization. Each acceptor inhibited the galactose transfer to the other one (up to 9573, presumably because both compete for the same enzyme.3. The transferase activities were enhanced by the nonionic detergent Triton X-100. The degree of activation depended directly on the amount of Triton bound to the membrane, i.e. the Triton/phospholipid ratio and not the wjv concentration of the detergent in the assay medium. This relationship persisted, regardless of the purity of the Golgi preparation : Half-maximal activation occurred at the same Triton/phospholipid ratio in postnuclear supernatants as well as in isolated Golgi fractions. The activation could not be explained by complete solubilization, because 50