Hypoxanthine formation from inosine

Fig. 7-1. Inosinate, the true end product of purine biosynthesis de novo in pigeon liver extracts incubated with substrates and C-formate. (A) Total radioactivity of products formed (B) specific activity of products formed. HX = hypoxanthine IMP-5 = inosinate. From (8). Reproduced with permission.

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. 

Slide 3 (Fig.l) shows that adenosine from the medium is rapidly converted to inosine,probably on the cell membrane, since no adenosine is detectable inside the cell. Inosine is rapidly converted to IMP in the normal cell,but in mutant cells it is converted to hypoxanthine. Effective AMP formation from adenosine in mutant cells is lower than in normal cells, indicating a shift towards hypoxanthine formation. 

The first surprise was that these molecules are much longer than seems necessary for the formation of adapters. In addition, 10-20% of their bases are modified greatly from their original form.171 Another surprise was that the anticodons are not all made up of "standard" bases. Thus, hypoxanthine (whose nucleoside is inosine) occurs in some anticodons. Conventional "cloverleaf" representations of tRNA, which display their secondary structures, are shown in Figs. 5-30 and 29-7. However, the molecules usually have an L shape rather than a cloverleaf form (Figs. 5-31 and 29-6),172 and the L form is essential for functioning in protein synthesis as indicated by X-ray and other data.173 Three-dimensional structures, now determined for several different tRNAs,174 175 are all very similar. Structures in solution are also thought to be... 

Figure 10.7 Schematic representation of the formation and fate of IMP. Formation of IMP is catalyzed by the enzyme hypoxanthine/guanine phosphoribosyl tranfer-ase (1) from the substrate hypoxanthine (Hypo) and phosphoribosyl pyrophosphate (PRibPP). IMP is shown undergoing several reactions the first (2) is catalyzed by 5 -nucleotidase to form inosine (INO) and orthophosphate (Pj) the other (3) is a two-step reaction catalyzed by sAMP synthetase to form adenylosuccinate (sAMP) and (4) by the enzyme sAMP lyase to convert sAMP to AMP and fumarate. Finally, (3) the deamination of AMP to IMP and NHa is catalyzed by AMP deaminase.

IMP v/hich was always the main labeled compound present within erythrocytes). After the lag time, the rate of hypoxanthine release was about the same of that observed in the absence of formycin B (fig. 2). Since, at the concentration employed, formycin B is known to inhibit purine nucleoside phosphorylase in intact human erythrocytes, these results confirm that the cells sequentially degrade the intracellular IMP to inosine and hypoxanthine and suggest that the phosphory-lase-catalyzed formation of hypoxanthine from its nucleoside is not the rate limiting step in this catabolic path. 

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. 

Fig. 8. The efiect of P5C on inosine monophosphate formation. Erythrocytes were incubated with 25 /iM [ C]hypoxanthine for 1 hour and nucleotides were separated using high-pressure liquid chromatography. The concentration of P5C was 0.5 mM. IMP pools in P5C-treated ( ) and control (O) cells as well as the incorporation of [ C]hypoxanthine into IMP in P5C-treated (A) and control (A) cells are shown. Taken from ref. (118).

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