Sialic acids radioactive precursors

There is some evidence for the occurrence of a glycolyl group at 0-4 of Neu5Ac in serum and submandibular-gland glycoproteins from the horse, based on biosynthetic studies with radioactive precursors,33 and chemical and t.l.c. analysis of the sialic acid ester groups.74... 

The pathway of the biosynthesis of Neu5Ac demonstrates the origin of sialic acids from the cellular hexose and hexosamine pools. These sugars are, therefore, suitable components for the study of the biosynthesis of sialic acid. However, only ManNAc has been shown to be a relatively specific precursor of sialic acids, as may be seen from the distribution of radioactivity between the individual monosaccharides of glycoconjugates after incubation. Injections of radioactive ManNAc into animals, or incubation of surviving tissue slices or individual cells with this compound, give incorporation of label mainly into the sialic acids.226 227... 

Radioactive acetate is a cheaper, readily available precursor for experiments on the labelling of sialic acid in tissues or cells, and it effectively labels the N- and O-acyl groups of sialic acids.228 This method is of great value not only for the preparation of radioactive sialic acids having high specific radioactivity but also for metabolic studies of sialic acids. However, the sialic acids must be isolated before determination of the specific radioactivity, as other aeetylated hexosamines are also labelled. 

When HeLa cells were.cultured in medium supplemented with 5 mM sodium butyrate, their content of GM3 increased (Fig.2a). Increases varied from 3.5 to 5-fold depending on the experiment (4,8,12,13). When the butyrate was removed and the cells were cultured in normal medium for 24 h, the GM3 levels returned to those found in untreated cells (Fig. 2a). Similar results were observed when N-[acetyl-3H]-D-mannosamine, a precursor of sialic acid, was also included in the culture medium. In the butyrate-treated cells, radioactivity associated with GM3 increased 6.5-fold and 24 h after butyrate was removed, the amount of labeling returned to control values (Fig. 2b). We also were able to label the GM3 by means of a cell surface labeling technique. Control and butyrate-treated cells were exposed to 10 mM sodium periodate and the oxidized sialyl residues were reduced with NaBSfy. There was 5.5-fold more 3h associated with the GM3 recovered from the butyrate-... 

The glutamine analog 6-diazo-5-oxo-L-norleucine (DON) inhibits L-glu-tamine n-fructose-6-phosphate amidotransferase (Section III) in the-pathway of conversion of C-glucose to both sialic acid and N-acetylga-lactosamine and, as expected, thereby inhibits incorporation of C-glu-cose into OSM. With glucose as the sole carbohydrate source in the incubation medium, DON also inhibits incorporation of radioactive amino acids into OSM but causes a concomitant increased incorporation of labeled amino acid into other protein fractions these latter proteins may contain nonglycosylated polypeptide precursors of OSM. All the inhibitory effects of DON are prevented if glucosamine is present in the incul ition medium. 

The use of [l-i CJglucosamine as a precursor of glycoprotein-bound sialic acid has been demonstrated in liver (e.g. Kohn et al. 1962, Lawford and Schachter 1966). Radioactivity was incorporated into microsomal glycoprotein (SARCiONEcr al. 1964), and the sialic acid label could be identified in Golgi fractions (Schachter 1974). However, glucosamine is converted into sialic acid and incorporated into... 

In the plasma membranes of rat liver the higher gangliosides GT ib, GD la, and GDib are degraded with a half-life of about 48 hrs, while GM3-bound sialic acid did not show any degradation at all (in the time-interval of 12 to 72 hrs after injection of the radioactive labeled precursor N-acetylmannosamine) (Hafermaas et al. 1982). Similar results have been obtained from studies on the turnover of plasma membrane glycoproteins (Kreisel et al. 1980, Tauber et al. 1982). This... 

Detection of glycopeptides is simplified if the samples can be labelled with radioisotopes. It is possible to metabolically label complex carbohydrates with isotopic precursors and these may provide some useful structural information. A glucosamine preeursor will specifically label N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc) and sialic acid whereas N-acetyl-mannosamine will only label sialic acid (19). Radioactive mannose is incorporated as mannose and fucose. A radiolabelling ratio of 3 1,... 

There is some indirect evidence that glycoproteins and sialic acids are found in mitochondria. When cells are grown in the presence of radioactive carbohydrate precursors, radioactivity can be found in isolated mitochondria (Molnar, 1967 Meezan, 1969 Wu, 1969 Buck et aL, 1974). Further, it has been shown that preparations of mitochondria from rat liver (Bosmann and Martin, 1969) contain transferases that add various sugars to glycoproteins, although transfer of sialic acid was not measured. The question of cross-contamination of mitochondria is as relevant in these studies as it is in direct chemical analysis for sialic acids. 

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