The biosynthesis of gangliosides. The incorporation of galactose, N-acetylgalactosamine and N-acetylneuraminic acid into endogenous acceptors of subcellular particles from rat brain in vitro

Abstract

Gangliosides bound to subcellular particles from rat brain were labelled by incubation of the particles (i) with CMP-N[(3)H]-acetylneuraminic acid and (ii) simultaneously, with CMP-N[(3)H]-acetylneuraminic acid and UDP-N-acetyl-[(14)C(1)]galactosamine or with CMP-N[(3)H]-acetylneuraminic acid and UDP-[U-(14)C]-galactose. Analysis of the labelled gangliosides showed that in (i), (a) the labelling was mostly in the neuraminidase-labile sialyl groups, (b) rigid relationships exist between the enzymes and the sialyl acceptors; the enzymes are not free to interact with all the specific substrates present in the preparation and (c) the precursor of the trisialoganglioside was the major disialoganglioside with a sialyl 2-->8 sialyl group. In (ii), (a) precursor-product relationships between the main pools of each ganglioside apparently do not exist, (b) for the labelling of Tay-Sachs ganglioside the amount formed from hematoside was at least 2.5 times that from aminoglycolipid and (c) the major monosialoganglioside was the precursor for the major disialoganglioside with a sialyl 2-->8 sialyl group.

The biosynthesis of gangliosides. Labelling of rat brain gangliosides in vivo

“…Gangliosides were extracted by the method of Folch, Lees & Sloane-Stanley (1957); the extracts were partitioned with water. The gangliosides were isolated from the upper phase and purified as described by Arce et al (1971). The specific radioactivity of each of the major gangliosides did not change after they were successively subjected to t.l.c.…”

“…with propanol-water (7:2.3, v/v) as solvent and passed through a column of Sephadex G-25 (Wells & Dittmer, 1963). The specific radioactivities of the neuraminidase-labile and -resistant NeuNAc from gangliosides were determined as previously described (Arce et al 1971).…”

“…Pathways for the synthesis of the carbohydrate chain of gangliosides have been worked out from experiments in vitro by using either exogenous or endogenous acceptors of glycosidic groups (Kaufman, Basu & Roseman, 1966, 1968; Arce, Maccioni & Caputto, 1966; Cumar, Fishman & Brady, 1971). Whereas there is general agreement among the investigators about the sugar nucleotide donors of those groups some minor discrepancies on the sequence of addition at the branching point still persist (for a review of the current situation see Arce et at. 1971).…”

Synthesis of Retinal Gangliosides During Chick Embryonic Development

“…In two of them the product chromatographed as two welldefined peaks, one of them running slightly ahead of GM, and the other slightly ahead of GM,. It is possible that the retinal homogenate contained endogenous UDP-Gal to convert a part of the synthesized GM, into GM, (see Arce et al, 1971;Cumar et al, 1971). In the third experiment, shown in Fig.…”

Ganglioside biosynthesis in Golgi apparatus: New perspectives on its mechanism

“…1) do not conform with results of ganglioside labeling in vivo. Labeling studies thus cast serious doubt whether any precursorproduct relationship really exists in vivo [Suzuki and Korey, 1964;Arce et al, 1971;Maccioni et al, 1971;Caputto et al, 19741. A lack of such a relationship was also observed in experiments in vitro with endogenous acceptors.…”