The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N-acetylmannosamine. I. Observations in hepatocytes.

Abstract

To study the site of incorporation of sialic acid residues into glycoproteins in hepatocytes, 40 g rats and 15 g Swiss albino mice were given a single i.v. injection of [3H]N-acetylmannosamine (8 mCi), and then sacrificed after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, 40 g rats were injected with 4 mCi of [3H]N-acetylmannosamine and sacrificed after 20 and 30 min, 1, 4 and 24 h, and 3 and 9 days. Concurrent biochemical experiments were carried out to test the specificity of injected [3H]N-acetylmannosamine as a precursor for sialic acid residues of glycoproteins. In radioautographs from rats and mice sacrificed 10 min after injection, grain counts showed that over 69% of the Ag grains occurred over the Golgi region. The majority of these grains were localized over the trans face of the Golgi stack, as well as over associated secretory vesicles and possibly GERL. In rats, the proportion of grains over the Golgi region decreased with time to 37% at 1 h, 11% at 4 h, and 6% at 24 h. The proportion of grains over the plasma membrane increased from 4% at 10 min to 29% at 1 h, and over 55% at 4 and 24 h; 2/3 of these grains lay over the sinusoidal membrane, and the remainder was equally divided over the lateral and bile canalicular membranes. Many Ag grains also appeared over lysosomes at the 4 and 24 h time intervals, accouting for 15-17% of the total. At 3 and 9 days after injection, light microscope radioautographs revealed a grain distribution similar to that seen at 24 h, with a progressive decrease in the intensity of labeling that by 9 days only a very light reaction remained. Because the biochemical findings indicated that [3H]N-acetylmannosamine is a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids), sialic acid is interpreted to be incorporated into these molecules in the Golgi apparatus; the latter then apparently migrate to secretion products, to the plasma membrane, and to lysosomes in a process of continuous renewal. Some of the label seen in lysosomes at later time intervals may have been derived from the plasma membrane or from material arising outside the cells.