Distribution of terminal glycosyltransferases in hepatic Golgi fractions.

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Abstract
The distribution of the 3 glycosyltransferases synthesizing the terminal trisaccharide sialic acid .fwdarw. D-galactose .fwdarw. N-acetylglucosamine present in many glycoproteins was determined in Golgi fractions [GF] prepared from rat liver homogenates. 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 3 morphologically and functionally different Golgi fractions GF1, GF2 and GF3 showed (in that order) decreasing specific activities of all 3 enzymes, but the relative amounts and relative specific activities of the 3 transferases in any given fraction were nearly identical. Two marginal fractions, one extra heavy (collected on the gradient below GF3) and the other extra light (isolated by flotation from the postmicrosomal supernate) contained recognizable Golgi elements. An enrichment of any transferase over the 2 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 EM), but failed to separate the transferases from each other; the 3 enzymes sedimented together with a population of empty vesicles to a density of .apprx. 1.08 g/ml.