Nucleotides fractionation

Fig. 10.1 (Continued), (c) Distribution of solutions within each local optimum for a basic amino acid dope (30% Arg, 30% Lys, 40% His). Each cluster consists of many different solutions, which are overlaid in die profile plots, revealing some trends. Each cluster emphasizes a different typical profile of solutions - and clusters 2 and 5 are especially different from die odier clusters. Comparing die mean values of each nucleodde between die clusters shows large differences, especially for die nucleotide fractions A1 and C3. The standard deviation for each nucleotide is 10%.

Nucleotide fractions appear in the boxes on the right side of the GUI. They can be entered manually or calculated by clicking the Compute by GALO button at the bottom of the GUI. In the current version of the GALO method [13], nucleotide fractions are calculated that are multiples of 1% of the total number of synthons in the mixture. Nucleotide fractions can be edited at any time. Click the Compute Manually button to recalculate the fractions of amino acids. 

A mutant of Escherichia coli B, resistant to 2,6-diaminopurine, was found to convert this base to 6-amino-2-methylamino-purine (XXVII) at a rate 60 to 90 % of that of normal cells. The methylated base was isolated from the culture medium, but the organism lacked the ability to incorporate 2,6-diaminopurine or its A-methyl derivative into the nucleotide fraction of the cell. This observation supports previous data which had indicated that resistance to this substance in Lactobacillus casei is due to the loss of an enzyme system responsible for the incorporation of the base into the nucleotide. ... 

Again, in the dystrophic mouse is incorporated twice as fast as in the normal animal into the acid-soluble phosphate of muscle (K7) indeed, by this means it is found that the turnover rate of some acid-soluble nucleotides is greater in dystrophic than in normal mice (ZIO). Recently it has been reported that the lives of dystrophic mice may even be prolonged by the administration of certain mixtures of purine and pyrimidine bases (Z9). In man it has been found that the nucleotide fraction... 

In general, thin layer methods are quicker and more sensitive than paper methods and in principle paper methods can be directly transferred to cellulose thin layers. In practice these methods have not been widely used for nucleotide fractionation (but see 1.2 and 3.4). The following manufacturers for example provide general information on the use of the products for thin layer chromatography H. Reeve Angel (Whatman) Camlab (Glass) Ltd Shandon Southern Ltd Eastman-Kodak. It is not proposed to go into more detail here. 

Fig. 2.1. Nucleotide fractionation. A sample of p2 phage RNA uniformly labelled with and the pyrimidines labelled with was digested with alkali ( 2.1.1) and separated by column chromatography on Dowex 50 formate eluted with 0.25 M ammonium formate pH 4.1. The ultraviolet transmission graph is traced from the record of a Uvicord I ultraviolet monitor output Radioactivities were measured by dissolving the samples in a scintillation cocktail and counting in a liquid scintillation counter. The peaks were identified from their ultraviolet spectra.

Uridine diphosphate A -acetyl-n-glucosamine has been isolated and structurally characterized. The compound can be obtained from a nucleotide fraction of yeast by elution from a Dowex-1 anion-exchange column, and from guineapig livers by precipitation as the barium salt. ... 

Additional purification of this relatively crude extract can be achieved by gel filtration on Sephadex G-50 equilibrated with 0.05 Af sodium acetate buffer (pH 3.8). Elution with the same buffer produces a large inactive protein fraction, followed by a smaller protein peak, and finally a nucleotide fraction. The hypocalcemic activity is associated with the descending limb of the second protein peak and the ascending limb of the nucleotide peak. The most active fractions are further purified by gradient elution with sodium acetate buffer on a carboxy-methyl-Sephadex G-25 column. Removal of the protein from the column is effected by gradient elution with sodium chloride in sodium acetate buffer. The hypocalcemic activity is associated with the descending limb of the protein peak. The authors claim that a 500-fold purification of thyrocalcitonin from the initial acid extract can be achieved. 

For individual nucleotides isolated by chromatographic procedures, the ultraviolet absorption spectra provide facile identification and quantitation (/). Well-known chemical (colorimetric) methods are available to identify and quantitate the sugar component, and to measure the relative amounts of sugar and phosphate in isolated nucleotide fractions (34). Enzymatic methods are also used in characterizing and in quantitation of nucleotides (35). 

In addition, nucleosides and nucleotides fractions were separated by development in saturated ammonium bicarbonate, and their sum total determined. 

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... 

This is good evidence for incorporation into (diH) [9R]Z by embryos and cotyledons, and into (diH) [9R]Z (derived from its 5 -nucleotides) by cotyledons. The nucleotide fraction was also degraded chemically, and was shown to be associated with (diH)Z. 

The bulk of the radioactivity in the hepatic venous effluent, after 5 minutes, was associated with the adenine, a smaller percentage of radioactivity with the nucleotide fraction, and still smaller amounts with the inosine plus xanthine, adenosine and hypoxanthine fractions. At the end of 20 minutes, there was a dramatic decline in percent radioactivity associated with the adenine fraction, and a concomitant rise in the radioactivity of the adenosine fraction. The other fractions exhibited less pronounced changes. 

Rabbit liver was perfused with phTj-hypoxanthine, as previously described, followed by washout perfusion for ten minutes with an isotonic solution of salts containing glucose, to remove extracellular label. An oxygenated washed human erythrocyte suspension was then perfused through the liver for 1 hr. Erythrocytes were collected and washed in an isotonic solution of salts. Liver and erythrocyte extracts were chromatographed and assayed. Purine bases, prepared by hydrolysis of the nucleotide fractions, separated by chromatography on Dowex 50. 

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