Nucleosides fractionation

Further degradation of the nucleosides to the purine bases by the catabolic activity of nucleoside phospho-rylase has not been detected in the assay system used. While the sum of the nucleotides and nucleo sides formed accurately reflects the enzyme activities, inhibition of the conversion of nucleotides to nucleosides by thymidine-5Hriphosphate (TTP) (5) facilitates the assay by measuring the nucleotides only. Indeed, for normal cells, the accumulation of radioactivity was found to be linear with time and protein concentration, in the nucleotide fraction only when TTP was present, while in the sum of the nucleotide and nucleoside fractions in both the presence and the absence of TTP. 

It is noteworthy that for the Lesch-Nyhan syndrome fibroblasts, the assay in the presence of TTP furnished the bulk of the radioactivity in the nucleoside fraction rather than in the nucleotide fraction, in contrast to normal cell extracts in which the bulk of the radioactivity is found in the nucleotide fraction. Two possible explanations may be entertained to explain this observation. First, in the HGPRT-deficient system the nucleotidase activity is far in excess over the HGPRT activity, and presumably the residual nucleotidase activity, not inhibited by TTP, is sufficient to degrade the relatively small amount of nucleotide formed. Secondly, the possibility should be considered that the accumulation of radioactivity in the nucleoside fraction reflects the anabolic activity of nucleoside phospho-rylase reacting the purine base with ribose-l-phosphate to form the nucleotide by an alternative pathway. However, this latter explanation seems to be invalid in view of the absence of the suitable substrate, ribose-l-phosphate, from the incubation system, and by the linearity... 

Polyamide is suitable for the separation of nucleic acid bases and nucleosides. It has been used in the analysis of cobra venom to detect the nucleoside fraction. The layer s great strength, in a host of solvent systems, is its ability to retain guanine. 

Methylcytosine (964 X = O) was synthesized in 1901 and its isolation from hydrolyzates of tubercule bacilli was reported in 1925. However, this was later shown to be incorrect and only about 1950 was it isolated by hydrolysis of the deoxyribonucleotide fractions from thymus, wheat germ and other sources (50MI21302). Nucleotides and a nucleoside of 5-methylcytosine are known. 

TBA)3-HP207 (0.68 g, 0.75 mmol) is added to the nucleoside tosylate (0.5 mmol) in MeCN (0.5 ml) and the solution is stirred at room temperature until the reaction is complete (monitored by NMR spectroscopy). H20 (l ml) is added and the solution is filtered through a 0.45-[xm Millex filter. The filtrate is purified by linear gradient elution from DEAE Fractogel (eluent 0.05-0.5 M aqueous NH4HC03) and the desired fractions are dried by lyophilization. 

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. 

In addition to classical reverse phase separation of peptides on octadecyl derivatized silica monoliths, sugars and peptides as well as proteins and nucleosides have been analyzed on a 20-cm-long silica-based poly(acrylic acid) column (ID. 200 pm), employing HILIC and weak cation-exchange chromatography, respectively [194]. Furthermore, HILIC fractionation of polysaccharides delivered remarkable and promising results [84,194]. 

F]-FLT is not or only marginally incorporated into DNA (<2%) and therefore not a direct measure of proliferation [122]. In vitro studies indicated that [ F]-FLT uptake is closely related to thymidine kinase 1 (TK1) activity and respective protein levels [117,118]. p F]-FLT is therefore considered to reflect TK1 activity and hence, S-phase fraction rather than DNA synthesis. Although being a poor substrate for type 1 equilibrative nucleoside transporters (ENT), cellular uptake of [ F]-FLT is further facilitated by redistribution of nucleoside transporters to the cellular membrane after inhibition of endogenous synthesis of thymidylate (TMP) de novo synthesis of TMP) [125]. However, the detailed uptake mechanism of [ F]-FLT is yet unknown and the influence of membrane transporters and various nucleoside metabolizing enzymes remains to be determined. 

The ion-exchange separation usually affords individual fractions of structurally related glycosyl esters of nucleoside pyrophosphates, containing the same nucleotide residue, but differing in the structure of the glycosyl groups. Separation of the esters of N-acetylhexos-amines, uronic acids, and neutral monosaccharides from one another is also usually achieved. 

Preparative, paper-chromatography is frequently used for further fractionation of the resulting mixtures. The high lability of glycosyl esters of nucleoside pyrophosphates seriously limits the choice of solvent systems. Systems used most commonly are neutral or slighdy acidic mixtures of ethanol with ammonium acetate,24,25 or weakly acidic solvents based on 2-methylpropionic acid.26 A solvent system containing morpholinium borate has also been found extremely useful.27... 

Ribonuclease U2 may be used for the synthesis of guanylyl-(3 -5 )-nucleoside and for the addition of adenylyl residue to 5 terminal of oligonucleotides including no guanylyl residue. By the fractionation of RNase U2 digests of RNA, oligonucleotides of defined sequence with 3 -terminal adenylic acid can be prepared. 

At least four acid phosphatase fractions have been obtained by ammonium sulfate, DEAE hydroxylapatite, and electrophoretic separation. One type of activity, nucleoside 2 - or 3 -phosphatase was purified 1500-fold. Hexosephosphatase activity was also obtained in three separate fractions. All three fractions were different with respect to rate of splitting of different substrates and pH optimum (118). 

Just as in the case of nucleosides, the older results were re-investigated with novel methods also for tritylation of pyranosides. All the monotrityl ethers of methyl p-D-xylopyranoside and methyl oc-D-xylopyranoside have been isolated recently [311] to complement the pioneering work by Hockett and Hudson [312], The monotrityl fraction obtained [313] from methyl a-L-fucopyranoside was now found [314] to consist of 2-0- and 3-0-trityl derivatives in the ratio of 3 2 (D-enantiomer has been actually studied). 

Under these conditions the octanucleotide linker fragments chromatograph at a similar rate to nucleoside triphosphates. There is usually sufficient of the labelled triphosphate, used for the 3 -end labelling, remaining in the sample to act as a marker for the linker peak which elutes later. The first fractions are enriched in the longer DNA fragments—which tend to clone with lower efficiency—and this crude fractionation decreases the number of random plaques which have to be picked to obtain cloned representatives of all the fragments. 

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