Enzymes labeled sugars

Sometimes, the most convenient method of preparation of the labeled sugar required is by chemical or enzymic modification of a more readily available, or commercially available, sugar. Thus, D-ribose-3-t has been prepared from l,2 5,6-di-0-isopropylidene-a-D-allofuranose-3-t by way of 1,2-O-isopropylidene-a-D-allofuranose-3-t, followed by periodate oxidation, and reduction.52... 

Many of the enzyme reactions previously discussed result in the incorporation of isotopes of hydrogen from the solvent or cofactor. Only those labeled compounds synthesized on a preparative scale are included in the Tables, but suitable modification of the other enzyme systems may make them suitable for use in the preparation of other labeled sugars. 

Synthesis. The synthases are present at the endomembrane system of the cell and have been isolated on membrane fractions prepared from the cells (5,6). The nucleoside diphosphate sugars which are used by the synthases are formed in the cytoplasm, and usually the epimerases and the other enzymes (e.g., dehydrogenases and decarboxylases) which interconvert them are also soluble and probably occur in the cytoplasm (14). Nevertheless some epimerases are membrane bound and this may be important for the regulation of the synthases which use the different epimers in a heteropolysaccharide. This is especially significant because the availability of the donor compounds at the site of the transglycosylases (the synthases) is of obvious importance for control of the synthesis. The synthases are located at the lumen side of the membrane and the nucleoside diphosphate sugars must therefore cross the membrane in order to take part in the reaction. Modulation of this transport mechanism is an obvious point for the control not only for the rate of synthesis but for the type of synthesis which occurs in the particular lumen of the membrane system. Obviously the synthase cannot function unless the donor molecule is transported to its active site and the transporters may only be present at certain regions within the endomembrane system. It has been observed that when intact cells are fed radioactive monosaccharides which will form and label polysaccharides, these cannot always be found at all the membrane sites within the cell where the synthase activities are known to occur (15). A possible reason for this difference may be the selection of precursors by the transport mechanism. 

The same procedure was applied212 for the preparation of uridine 5 -(a-D-glucopyranosyl pyrophosphate-4"-t, or -5"-f. A cell-free extract of Phytophthora cinnamoni was used for the synthesis of the 6"-t derivative,213 as well as yeast pyrophosphorylase.2133 An enzyme from Salmonella typhimurium was found quite satisfactory214 for obtaining this and other labeled nucleotide sugars. ... 

---