Inosine from hypoxanthine

The existence of a separate phosphoribosyltransferase for adenine was first deduced because gentle heating of crude enz3rme preparations destroyed this activity, without decreasing their ability to make inosinate from hypoxanthine and PP-ribose-P. Adenine phosphoribo ltransferases have since been separated from other purine phosphoribosyltransferases by ion-exchange chromatography, electrophoresis, and gel filtration, and the separate identities of these enzymes have also been demonstrated by specific mutations, both in man and in bacteria. 

Adenosine is formed from ATP via a phosphatase cascade that sequentially involves the diphosphate, ADP, and the monophosphate, AMP. The actions of adenosine are terminated by uptake and rephosphorylation via adenosine kinase to AMP or by cataboHsm via adenosine deaminase to inosine and hypoxanthine. 

The decrease in the concentration of IMP preceded a simultaneous increase in the concentrations of both inosine and hypoxanthine in meat samples. (From Tikk et ah, 2006)... 

The nucleoside formed from hypoxanthine and ribose is known as inosine (Ino or I) and the corresponding nucleotide as inosinic acid. Further substitution at C-2 of -H by -OH and tautomerization yields xanthine (Xan). Its nucleoside is xanthosine (Xao, X). A similar hydroxylation at C-7 converts xanthine to uric acid, an important human urinary excretion product derived from nucleic acid bases. 

Figure 9.99 Separation of the components of the reaction studied (catalysis by nucleoside phosphorylase of a purine riboside-base conversion) by HPLC. Chromatographic conditions isocratic elution flow rate, 2 mL/min 0.02 F KH2P04 (pH 4.2) and 3% methanol. Peaks 1, uric acid 2, hypoxanthine 3, xanthine 4, inosine. (From Halfpenny and Brown, 1980.)...

HGPRT Hypoxanthine-guanine phosphoribosyltransferase the enzyme that catalyzes the synthesis of inosine monophosphate (IMP) and guano-sine monophosphate (GMP) from hypoxanthine and guanine, respectively. It makes up part of the purine salvage pathway, a way of recycling purine bases back to the nucleotides. 

Maire, J., MedUanski, J., and Straub, R., Release of adenosine, inosine, and hypoxanthine from rabbit non-myehnated nerve fibers at rest and during activity, J. Physiol, 357,67,1984. 

Inosinic Acid, 5 -Inosinic acid 5-tnosimc acid muscle inosinic acid t-inosinic acid hypoxanthine ribosjde-5-phosphoric acid IMP. C.jH.jNjOjP mol wt 348.22. C 34.49%, H 3,76%, N 16.09%, O 36,76%, P 8,90%. Prepn from meat extract Levene, Bass, Nucleic Acids (New York, 1931) p 229 from dried sardines Yoshida, Kageyama,... 

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.

The radioactivity lost from the adenine nucleotide pool is recovered in various purine derivatives. There is a transient increase in radioactive inosine and hypoxanthine, and a nearly constant accumulation of radioactive allantoin (Fig. IB). There is also a transient accumulation of radioactive adenosine and IMP (data not shown). However, the extent of that accumulation depends upon the moment that the cells are exposed to glycerol. When glycerol is added at the beginning of the incubation, about 8% of the total radioactivity is found both in adenosine and in IMP. However, when glycerol is added after a 15 min incubation, 17% of the total radioactivity is found in IMP and only 3% in adenosine. All these changes are similar to those induced by fructose under identical conditions (data not shown). 

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. 

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. 

The susceptibilities of some of these fluorinated purine nucleosides to the action of enzymes are now described. In contrast to the inertness of the 2 -deoxy-2 -fluoro- and 3 -deoxy-3 -fluorocytidine analogs 739, 744, and 821 towards cytidine deaminase, the adenosine compounds 867, 883, and 906 are readily deaminated - by the adenosine deaminase in erythrocytes and calf intestine, but the resulting (deaminated) inosine compounds (from 867 and 883), as well as 888, are highly resistant - to cleavage by purine nucleoside phosphorylase (to give hypoxanthine base for the first two). The reason was discussed. Both 867 and 883 can form the 5 -triphosphates, without deamination, in human erythrocytes or murine sarcoma cells in the presence of 2 -deoxycoformycin, an adenosine deaminase inhibitor, but... 

Figure 12 Gradient separation of bases, nucleosides and nucleoside mono- and polyphosphates. Column 0.6 x 45 cm. Aminex A-14 (20 3 p) in the chloride form. Eluent 0.1 M 2-methyl-2-amino-l-propanol delivered in a gradient from pH 9.9-100 mM NaCl to pH 10.0-400 mM NaCl. Flow rate 100 ml/hr. Temperature 55°C. Detection UV at 254 nm. Abbreviations (Cyt) cytosine, (Cyd) cytidine, (Ado) adenosine, (Urd) uridine, (Thyd) thymidine, (Ura) uracil, (CMP) cytidine monophosphate, (Gua) guanine, (Guo) guanosine, (Xan) xanthine, (Hyp) hypoxanthine, (Ino) inosine, (Ade) adenosine, (UMP) uridine monophosphate, (CDP) cytidine diphosphate, (AMP) adenosine monophosphate, (GMP) guanosine monophosphate, (IMP) inosine monophosphate, (CTP) cytidine triphosphate, (ADP) adenosine diphosphate, (UDP) uridine monophosphate, (GDP) guanosine diphosphate, (UTP) uridine triphosphate, (ATP) adenosine triphosphate, (GTP), guanosine triphosphate. (Reproduced with permission of Elsevier Science from Floridi, A., Palmerini, C. A., and Fini, C., /. Chromatogr., 138, 203, 1977.)...

Phillis, J. W., O Regan, M. H. Walter, G. A. (1988). Effects of deoxycoformycin on adenosine, inosine, hypoxanthine, xanthine, and uric acid release from the hypoxemic rat cerebral cortex. J. Cereb. Blood Flow Metab. 8 (5), 733-41. 

Name changes may be confusing here when AMP loses the phosphate to become adenosine and adenosine loses the ribose to become adenine, it s still easy to tell who came from where. When IMP loses the phosphate, it becomes inosine, but when inosine loses the ribose it becomes hypoxanthine. It may be a little confusing, but it s still better than trying to pronounce inonine. 

In the most important degradative pathway for adenosine monophosphate (AMP), it is the nucleotide that deaminated, and inosine monophosphate (IMP) arises. In the same way as in GMP, the purine base hypoxanthine is released from IMP. A single enzyme, xanthine oxidase [3], then both converts hypoxanthine into xanthine and xanthine into uric acid. An 0X0 group is introduced into the substrate in each of these reaction steps. The oxo group is derived from molecular oxygen another reaction product is hydrogen peroxide (H2O2), which is toxic and has to be removed by peroxidases. 

The major intermediates in the biosynthesis of nucleic acid components are the mononucleotides uridine monophosphate (UMP) in the pyrimidine series and inosine monophosphate (IMP, base hypoxanthine) in the purines. The synthetic pathways for pyrimidines and purines are fundamentally different. For the pyrimidines, the pyrimidine ring is first constructed and then linked to ribose 5 -phosphate to form a nucleotide. By contrast, synthesis of the purines starts directly from ribose 5 -phosphate. The ring is then built up step by step on this carrier molecule. 

Thymine, hypoxanthine, xanthine, uridine, thymidine, adenine, inosine, adenosine, and guanosine are purchased from Calbiochem. Orotic acid, pseudouridine,... 

An amino group is removed from AMP to produce IMP, or from adenosine to produce inosine (hypoxanthine-ribose) by AMP or adenosine deaminase. 

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... 

Position of Bond Cleavage PNP (EC 2.U.2.1) from human erythrocytes (homogeneous, purified by formycin B affinity chromatography) as well as from E, coli were allowed to equilibrate a mixture of R-l-[1 0lj]-P, pl Oij, hypoxanthine and inosine at pH 7-00 in 10 mm NMR tubes. The chemical shift differences of the 31p nuclei of the two R-l-P s (13 9 Hz for the human erythrocytic and 13.1 Hz for the E. coli enzyme) as well as of the two P3 resonances (13 9 Hz for erythrocytic and 13-7 Hz for E. coli source) clearly indicated C-0 bond cleavage by these enzymes as well. In addition, no evidence was found over the time course of the NMR measurements (l hr) for purine nucleoside phosphorylase catalyzed exchange of pl o + H2O (solvent) J randomized P. Therefore,... 

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