Desoxyribose-5-phosphate

F9. Friedkin, M., and Kalckar, H. M., Desoxyribose-1-phosphate. I. The phosphoroly-sis and resynthesis of purine desoxyribose nucleoside. J. Biol. Chem. 184, 437-449 (1950). 

An enzyme, termed desoxyribose phosphate aldolase, - has been found widely distributed in microorganisms and animal tissues. The enzyme catalyzes the following reversible reaction ... 

This product appeared identical with the desoxyribose phosphate liberated from desoxyadenylic acid. 

These reactions have been described by Friedkin and Kalckar and by Manson and Lampen - the desoxynucleoside phosphorylase will be discussed in greater detail in the chapter, Nucleotides and Nucleosides. Desoxyribose-5-phosphate has been generated from the nucleoside, and Hoffmann and Lampen have proceeded to the study of the degradation of desoxyribose phosphate in E. 

RNA is synthesized in the latter case, and since the DNA synthesis is stimulated about fourfold, there appears to be a shunt of ribose phosphate synthesis to desoxyribose phosphate synthesis in virus infection. Since nucleic acid P accounts for about 80% of the phosphorus of the cell, it is evident that this redistribution of pentose P involves a major redistribution of phosphorus metabolism. 

As shown above, a shunt does occur in the mode of the utilization of glucose-6-phosphate in the shift from normal growth to virus synthesis. We can exclude two possible mechanisms. First, the change in glucose utilization to the glycolytic pathway is not merely a shift of reversible systems caused by removal of desoxyribose phosphate from the system by virus. In variations of the basic virus-host system, RNA synthesis is completely inhibited even though DNA synthesis may be diminished or even completely repressed. 

The furanose form of D-ribose is required for the enzymatic phosphorolysis reaction. Pyranose-ribose-l-phosphate will not react with hypoxanthine to liberate inorganic phosphate. On the other hand, desoxyribose nucleosides are quite reactive ... 

Desoxyribose-l-phosphate has been obtained as the crystalline cyclo-hexylamine salt. It is even more acid-labile than ribose-l-phosphate. Thus, 50% of the phosphorus is released as inorganic phosphate within 10 to 15 minutes upon hydrolysis at pH 4, at 23°C. The equilibrium favors synthesis. It is felt that the same enzyme catalyzes the phosphorolysis of hypoxanthine riboside and hypoxanthine desoxyriboside, because the Michaelis-Menten constants are nearly identical and there is no summation of rates with both substrates present at optimal concentrations. Furthermore, nucleoside phosphorylase purified from horse liver is active with both nucleosides and desoxynucleosides. ... 

The ribose phosphate normally present in cells is jS-D-ribose- 1-phosphate it is very add-labile. Desoxyribose-l-phosphate is even more unstable in add solution at pH 4-0 at room temperature, it is 50% hydrolysed in 15 minutes. It is hydrolysed in the course of estimations of inorganic phosphate and is consequently often measured as such. 

Two methods, the one based on the alkali lability of RNA and the other on the acid lability of both RNA and DNA, appeared simultaneously in 1945 and have provided the analytical foundation for much of the recent research activity revolving about nucleic acids. The former of these methods is that of Schmidt and Thannhauser (abbreviated here as ST) (91) which depends upon the selective conversion of RNA phosphorus to organic acid-soluble phosphorus (mononucleotides) by alkali after removal of acid-soluble phosphates and of phospholipid phosphorus (16 et ante). The method of Schneider (S) (93) extracts the same lipid-free protein precipitate with hot trichloroacetic or perchloric (95) acid which solubilizes both RNA and DNA the estimation of each in the mixture utilizes specific colorimetric reactions for the (purine) ribose and desoxyribose moieties. These two methods will be discussed in connection with the combined method which follows. 

Step 3. Alkali dissolves most of the RNA-DNA-protein residue and hydrolyzes RNA quantitatively to mononucleotides (91). All four of these contain phosphorus but only the purine nucleotides react in the conventional ribose assay. Alkali also releases phosphoprotein phosphorus as inorganic phosphate (91) (the degree of completeness has not been reported). DNA is alkali resistant as long as the purine-desoxyribose linkages are intact (these bonds are acid labile—see comments under step 1). Once adenine and guanine are removed, alkali lability of DNA appears, due presumably to a cyclization mechanism similar to that which is responsible for RNA lability (14). 

Step 4. Treatment with hot acid releases all the adenine and guanine (99) and some desoxyribose and inorganic phosphate from DNA, the remainder appearing as acid-soluble pyrimidine-nucleotide combinations (36). Diphenylamine reacts only with the free desoxyribose or purine de-soxyribosides. Orcinol reacts slightly with DNA hence its use for RNA assay in the presence of large amounts of DNA requires a correction (93). 

The enzyme has been purified from extracts of E. coli and is optimally active between pH 6 and 8. No evidence for the existence of a coenzyme could be obtained. With a purified enzyme preparation, the reactive triose phosphate was seen to be glyceraldehyde-3-phosphate rather than dihy-droxyacetone phosphate. Free desoxyribose or trioses were unreactive in the system. The enzyme was inhibited by chloral hydrate and propion-aldehyde, albeit at relatively high concentrations. 

It is of interest that extracts of E. coli can convert ribose-5-phosphate to desoxyribose-5-pho hate in the presence of added acetaldehyde. This conversion presumably passes throu libulosOnS-phosphate. 

The desoxyribose-5-phosphate generated enzymatically as described above has been converted to hypoxanthine desoxyriboside in extracts of E. coli in two reactions, analogous to the phosphoribomutase and nucleoside phosphorylase reactions. These are ... 

Under conditions of virus infection with the same CO2 production per mole of glucose, 29% of the Ci was contained in the CO2. This is equivalent to a maYinmiTTi of 29% of the glucose metabolized via the oxidative pathway or a minimum of 6%. These differences were real and reproducible and are of the same order as the shifts in the amounts of pentose and desoxypentose synthesized during glucose utilization. It was suggested as a result of these studies that the ribose of RNA was derived mainly via the oxidative pathway, whereas the desoxyribose of DNA arose from triose phosphate generated from the Embden-Meyerhof scheme. 

After administration of labeled phosphate to mice, the content of nucleoproteins (containing both desoxyribose and ribose compounds) of... 

Kohman and Rusch (109) administered labeled phosphate to rats and mice and determined the content of nucleoproteins (containing both desoxyribose and ribose compounds) of normal liver and of liver in which cancer was produced by feeding azo dyes. The tumorous liver, in which a rapid formation of new cells takes place, was found to have a 45% increase in uptake of compared with the normal liver. 

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