Ganglioside biosynthesis in rat liver. Characterization of UDPgalactose- glucosylceramide galactosyltransferase and UDPgalactose-GM2 galactosyltransferase

Abstract

The conditions for the quantitative determination of UDP-Gal :glucosylceramide galactosyltrai~sferase and of UDP-Gal : GM, galactosyltransferase in Golgi-enriched preparations of rat liver were optimized. Triton X-100 was the detergent routinely used as octyl glucoside acted as a galactose acceptor forming octyl lactoside. Manganese ions were required for full activity, but Co2+ and Mg2+ could substitute to some extent. The nucleotide pyrophosphatase activity of the Golgi preparations which interfered with the GL,-synthase assay was inhibited by addition of 20 rnM IMP; the latter is without appreciable effect on the rate of GL, synthesis. Apparent K, values for UDP-Gal were 130 pM and 140 pM with GI,-synthase and Gm,-synthase, respectively. That for glucosylceramide was 80 pM with GL,-synthasc; for GM, it was 10 pM with GM,-synthase. Competition experiments with variable concentrations of the lipid acceptors showed that the two synthase activities are independent catalytic entities. The specific activity of GM,-synthase exceeds that of GL,-synthase by a factor of ca. 25 under the optimized conditions used here.Gangliosides are a group of acidic glycosphingolipids mainly found in plasma membranes of neural and extraneural organs [I]. Several receptor functions were attributed to this group of lipids, but only GM, has been established so far as receptor in the case of cholera toxin [2] and, possibly, of interferon [3]. The biosynthesis of gangliosides in rat liver occurs predominantly in the Golgi apparatus [4]. Monosaccharides and sialic acids are added to the appropriate glycolipid acceptors in ordered sequence by transferases that are membrane-bound and probably organized in a multienzyine complex [S,h].Previous investigations in this laboratory showed a strong inhibition in vivo of G M I and GD,,,, biosynthesis in the liver of rats previously injected with D-galactosamine [7]. This aminosugar leads to a change in the hepatic UDP-sugar levels with lowered content of UDP-Glc and UDP-Gal and elevated content of UDP-hexosamines and UDP-N-acetylhexosamines. Ganglioside biosynthesis could, therefore, be impaired by the

Ganglioside biosynthesis in Golgi apparatus: New perspectives on its mechanism

“…That this is not true was shown by MorrC and associates Keenan, 1974;Wilkinson et al, 19761, who, while studying with rat liver and cow mammary gland, found that the glycosphingolipid glycosyltransferases, the enzymes that synthesize the gangliosides, were mainly localized in the Golgi apparatus, where the enzyme enrichments were up to 50 times more over the starting homogenate; the plasma membrane preparation of the respective tissues, on the other hand, had very little of these enzyme activities. Many subsequent studies have established this, and almost all the glycosyltransferases of the rat liver Golgi apparatus have been studied and partially characterized [Wilkinson et al, 1976;Richardson et al, 1977;Eppler et al, 1980;Senn et al, 1981Senn et al, , 1983Kaplan and Hechtman, 19831. It is now believed that the gangliosides, after being synthesized in the Golgi apparatus, are transported to the plasma membrane, from where they reach the lysosomes for breakdown by the degrading enzymes.…”

Localization in the Golgi apparatus of rat liver UDP-Gal:glucosylceramide .beta.1.fwdarw.4galactosyltransferase

“…After its first discovery in the nervous system (Basu et al, 1968) and in the rat spleen (Hildebrand & Hauser, 1969), it was reported in other tissues such as bone marrow (Taki et al, 1982) and renal cells (Chatterjee & Castiglione, 1987). In rat liver, the presence of GalT-2 was suggested (Senn et al, 1983), but exogenous GlcCer-dependent formation of true LacCer could not be demonstrated in vitro (Walter et al, 1983). In vivo, the occurrence of glycosylation of exogenous GlcCer (Trinchera et al, 1990b) suggests that GalT-2 fThis work was supporlcd by a grant from the Ministero della Pubblica Islruzione, Rome (Grant MPI 1989, 60%).…”

Both GA2, GM2, and GD2 synthases and GM1b, GD1a, and GT1b synthases are single enzymes in Golgi vesicles from rat liver.

“…11). These reactions are catalyzed by specific glycosyltransferases, many of which were studied and partially characterized in rat liver Golgi apparatus (12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Most major mammalian gangliosides derive from the a or b series (22).…”