Hypoxanthine incorporation into nucleic acid purines

Adenine is inert in the nucleoside phosphorylase systems of both mammalian tissues and microorganisms, but isotopically labeled adenine is effectively incorporated into nucleic acid purines, both in rats " and in yeast.This poses a question as to the possible role of nucleoside phosphorylase in polynucleotide synthesis. It has been suggested that hypoxanthine or guanine nucleosides (or nucleotides) are synthesized first. Then an exchange reaction with free adenine (or a derivative) might occur, For example, adenine might react with inosine to form adenosine and hypoxanthine. Some known exchange reactions are discussed below. 

Dietary purines are not an important source of uric acid. Quantitatively important amounts of purine are formed from amino acids, formate, and carbon dioxide in the body. Those purine ribonucleotides not incorporated into nucleic acids and derived from nucleic acid degradation are converted to xanthine or hypoxanthine and oxidized to uric acid (Figure 36-7). Allopurinol inhibits this last step, resulting in a fall in the plasma urate level and a decrease in the size of the urate pool. The more soluble xanthine and hypoxanthine are increased. 

Allopurinol facilitates the incorporation of xanthine and hypoxanthine into nucleic acids, lowering the substrates for catabolism. In addition, the decrease in xanthine metabolism facilitates a negative feedback on purine synthesis. 

Hypoxanthine, on the other hand, which accounts for only a fifth or so of the urinary uric acid is an active intermediate. It is degraded to xanthine and then to uric add by xanthine oxidase. This enzyme is found mainly in liver, kidney, and bowel, while guanase is widely distributed and would quickly deaminate any guanine formed. The product xanthine is a poor substrate for hypoxanthine phosphoribosyltrans-ferase (HPRT). Most of the hypoxanthine formed is reutiliiced by conversion to inosinic acid. Similar conclusions were reached by Ayvazian and Skupp in 1965 when they administered C-labeled purines to patients (A2). Furthermore, these studies and those earlier studies show that the xanthine is converted to hypoxanthine, presumably at the nucleotide level, and on the basis of what we know about microorganisms, we would assume it to be via guanine nucleotides (M2). Since label was found in urinary 7-methylguanine as early as 4 hours after administration of C-labeled purines, and since methylation of RNA occurs at the macromolecular level (B13), interconversion must be rapid and incorporation of some of these products into nucleic acids must also occur quickly. 

Table 2 shows the effect of each purine inhibitor on incorporation of ( H) hypoxanthine into nucleic acids (acid-insoluble PCA fraction) of malaria infected erythrocytes. Two clinically proven antimalarial drugs, chloroquine (7-chloro-4-(4 -diethylamino-methyl-butylamino)-quinoline) and mefloquine (a-(2-piperidyl)-2, 8-bis (trifluoro-methyl)-4-quinolinemethenol hydrochloride), were included for comparison. Hadacidin, bredinin and mycophenolic acid all produced significant (p <. 005) decreases in nucleic acid synthesis by PRBC as measured by incorporation of ( H) hypoxanthine. Alanosine had no demonstrable effect. 

Autoradiography with labeled substrates on cultured fibroblasts has been used to visualize HG-PRT deficiency cells of deficient individuals lack the ability to incorporate hypoxanthine or guanine into nucleic acids, because these purine bases can not be converted to their corresponding mononucleotides (1,2). The alternative pathway to form IMP or GMP via inosine or guanosine is not likely, for, although nucleoside phosphorylase is present, there is no definite evidence for the existence of inosine- or guanosine kinase in human cells (3,4). 

The potential utility of N-labelcd oligonucleotides to probe unique nucleic acid structure, drug binding, and nucleic acid- protein interactions has led to considerable interest in the development of routes to the requisite N-labeled monomers. In the purine series, chemically synthesized N 1-labeled hypoxanthine was incorporated into a yeast tRNA by fermentation and... 

It is also possible that the high xanthine oxidase activity of rat tissues was responsible for the nonutilization of hypoxanthine, since the base was extensively converted to allantoin (800). However, it is well known that hypoxanthine, as well as other purines, were also substrates for an anabolic enzyme system in the rat, namely, nucleoside phosphorylase (816). Hypoxanthine was readily utilized for the synthesis of both adenine and guanine of RNA and DNA in slices of rabbit bone marrow (808a), even though it was not incorporated into the nucleic acids of the rat (800). Bone marrow may lack xanthine oxidase, thus leaving hypoxanthine available to participate in synthetic reactions. Thus, the competition of several enzyme systems for the administered compound may have determined its final disposition. 

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