Hypoxanthine inosinic acid formation from

When RNA of tobacco mosaic virus is incubated with nitrous acid, changes occur in the base composition because nitrous acid deaminates cytosine and adenine. (It also deaminates guanine, but not in the intact virus.) Cytosine is converted to uracil and adenine is converted to hypoxanthine (adenylic acid inosinic acid). Consequently, the six amino groups of adenine and cytosine are replaced by keto groups. Since the complementariness of the newly synthesized RNA chain is dictated by the formation of hydrogen bonds with the bases in the template, the newly synthesized RNA is different from the wild RNA and thus a base mutation has been introduced experimentally. 

Amino-4-imidazole carboxamide ribotide, a precursor only two steps removed (formylation and cycli-zation) from inosinic acid, can be synthesized by the direct condensation of the imidazole with 5-phosphori-bosyl pyrophosphate. The enzyme catalyzing this reaction was purified from an acetone powder of beef liver. The same enzyme (AMP pyrophosphorylase) catalyzes the condensation of adenine, guanine, and hypoxan-thine. Nucleoside phosphorylase is an enzyme that catalyzes the formation of a ribose nucleoside from a purine base and ribose-1-phosphate. Guanine, adenine, xanthine, hypoxanthine, 2,6-diaminopurine, and aminoimidazole carboxamide are known to be converted to their respective nucleosides by such a mechanism. In the presence of a specific kinase and ATP, the nucleoside is then phosphorylated to the corresponding nucleotide. 

Tissues were homogenized in phosphate buffer and sonicated, and the supernatant was used for electrophoresis. Samples were run in phosphate buffer, pH 8.5, on cellulose acetate paper for 2 hours at 4 and at 0.5 mA/cm. PRPP synthetase was located on the paper by a radiochemical assay formation of PRPP from ribose-5-phosphate and ATP was coupled to inosinic acid (IMP) synthesis by the addition to the reaction mixture of labelled hypoxanthine and partially purified hypoxan-thine-guanine phosphoribosyltransferase (HGPRT). 

Similar results by Schulman and Buchanan indicated that inosinic acid was an intermediate in the synthesis of hypoxanthine from labeled glycine. Although the reactions of inosinic acid and formate are more involved than originally thought (infra vide), there is agreement that Buchanan, J. M., and Schulman, M. P., J. Biol. Chem. 202, 241 (1953). 

In order to observe regularly the enzymatic exchange reaction, it was necessary to add inosinic acid to the extract and limit de novo synthesis by omitting bicarbonate from the system. When the incubation was carried out in the absence of both bicarbonate and added inosinic acid, labeled glycine and formate were converted into inosinic acid in the ratio expected from de novo synthetic reactions. Inosine and hypoxanthine could not replace inosinic acid, thus demonstrating that the latter was the specific substrate in the enzymatic exchange reaction. 

A reaction similar in type to that described above has been demonstrated in liver extracts by Wajzer and Baron for inosine-3 -phosphate synthesis from hypoxanthine and ribose-3-phosphate. The formation of the mononucleotide, adenylic acid, by the phosphorylation of adenosine by adenosinetriphosphate has also been described. The significance and integration of these different reactions remains a major problem for future effort. 

---