Inosine phosphorylase

Inosine can be converted directly to inosinate by inosine kinase. This enzyme has been shown to exist in human cells, but at a low level (P2). It undoubtedly does not play a major role. Inosine phosphorylase cleaves inosine to hypoxanthine and ribose I-phosphate (Kl). Inosine is formed by the deamination of adenosine catalyzed by adenosine deaminase, an enzyme found in varying concentrations in essentially all normal mammalian cells examined. Studies with an inhibitor of adenosine deaminase, ribosyl-4-amino-5-imidazole carboxamide, on several strains of Escherichia coli revealed a major role for the enzyme. In cells with a block in purine synthesis a condition of guanine deprivation occurred after growth with adenine as purine source, and there was a derepression of the enzymes that convert IMP to XMP and XMP to GMP (K17). 

The patient s RBC ADA activity was 43 000 nmol.min . ml RBC " (normal values 495 i 60), There was an about 3-fold increase of red cell pyrimidine 5 -nucleotidase and orotate phosphoribosyl-transferase, whereas other enzymes of purine and pyrimidine metabolism (inosine phosphorylase, adenosine kinase, adenine phosphoribo-syltransferase, hypoxanthine-guanine-phosphoribosyltransferase, phosphoribosylpyrophosphate synthetase) were normal or slightly elevated. There was a 6-fold increase of pyruvate kinase activity relatively to comparably reticulocyte-rich blood, and a 1.5 to 3-fold increase of the other enzymatic activities of glucose and glutathione metabolism. Plasma ADA was much elevated (30.5 pmol.min . ml normal value 5.1 - 2.5), probably reflecting intravascular hemolysis. ADA activity in lymphocytes (2.13 nmol.min 1.10 cells normal 1.93 0.61) and in fibroblasts (26 nmol.min l.mg protein 1 normal range 14-118) was normal, whereas the small increase of activity in platelets (59.5 nmol.min . 10 cells control 26.7) and in the liver (8.4 pmol.min . mg protein" normal ... 

Adenosine Kinase AMP Kinase ADP Kinase Adenosine Deaminase Inosine Phosphorylase... 

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 3.29 Control of an enzyme activity by multiple allosteric regulators. The enzyme glycogen phosphorylase b in muscle is regulated by changes in the concentrations of AMP and inosine monophosphate (IMP) (which are activators) and ATP and glucose 6-phosphate (G6P), which are inhibitors.

Phosphorylases remove the ribose sugars to yield the bases guanine or hypoxan-thine (from adenine or inosine nucleosides). 

Arsenate similarly replaces phosphate in various phosphorolysis reactions, so that sucrose phosphorylase catalyzes the hydrolysis of sucrose in its presence (23), potato phosphorylase can hydrolyze amylose and amylopectin (24), nucleoside phosphorylase can hydrolyze inosine... 

Purine nucleoside phosphorylase converts inosine and guanosine into their respective purine bases, hypoxanthine and guanine. 

The conversions of inosine to hypoxanthine (Fig. 25-17, step e), of guanosine to guanine (step g), and of other purine ribonucleosides and deoxyribonucleo-sides to free purine bases are catalyzed by purine nucleoside phosphorylase.318 321b Absence of this enzyme also causes a severe immune deficiency which involves the T cells. However, B cell function is not impaired.312 315 322... 

After purine nucleotides have been converted to the corresponding nucleosides by 5 -nucleotidases and by phosphatases, inosine and guanosine are readily cleaved to the nucleobase and ribose-1-phosphate by the widely distributed purine nucleoside phosphorylase. The corresponding deoxynucleosides yield deoxyribose- 1-phosphate and base with the phosphorylase from most sources. Adenosine and deoxyadenosine are not attacked by the phosphorylase of mammalian tissue, but much AMP is converted to IMP by an aminohydrolase (deaminase), which is very active in muscle and other tissues (fig. 23.20). An inherited deficiency of purine nucleoside phosphorylase is associated with a deficiency in the cellular type of immunity. 

HX), inosine Catla nucleoside phosphorylase enzyme Pt electrodes/0.7V (FCA)... 

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,... 

A close look at this reaction reveals that in the opposite direction, the reaction is of the phosphorolysis type. For this reason, the enzymes catalyzing the reaction with ribose-l-phosphate are called phosphorylases, and they also participate in nucleic acid degradation pathways. Purine nucleoside phosphorylases thus convert hypoxanthine and guanine to either inosine and guanosine if ribose-l-phosphate is the substrate or to deoxyinosine and deoxyguanosine if deoxyribose-1-phosphate is the substrate. Uridine phosphorylase converts uracil to uridine in the presence of ribose-l-phosphate, and thymidine is formed from thymine and deoxyribose-l-phosphate through the action of thymidine phosphorylase. 

Adenosine deaminase catalyzes the deamination of adenosine to form inosine and ammonia. The inosine (Ino) can be degraded further to hypoxanthine (Hyp) by nucleoside phosphorylase, an activity often present in extracts. Therefore, in many cases, the assay involves a determination of either the loss of adenosine (Ado) or the formation of both inosine and hypoxanthine. An early study by Uberti et al., 1977, was followed by another by Hartwick et al., 1978. 

The activity was obtained from a lysate of red blood cells. The reactions were terminated with a boiling water bath (45 s), and the samples clarified by centrifugation. Samples of 5 fiL were analyzed. Figure 9.94B, C, D shows the chromatographic profiles obtained after incubation times of 3,30, and 50 minutes, respectively, with the enzyme. The loss of adenosine is noted, but the effect of the nucleoside phosphorylase is seen, since hypoxanthine, not inosine, is the final product. 

Nucleoside phosphorylase catalyzes the reversible conversion of a purine riboside such as inosine to a purine base such as hypoxanthine and ribose-1-phosphate. Free phosphate is also required as a substrate. 

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.)...

Inosine Phosphate Purine nucleoside phosphorylase (EC 2.4.2.1) 7.4 Hypoxanthine, uric acid... 

Finally, the serum sample was incubated with the enzyme purine nucleoside phosphorylase and rechromatographed (Fig. 14C). From the disappearance of the inosine peak and the appearance of a peak with the retention time of hypoxanthine, it can be concluded that the peak under investigation was indeed inosine. 

Fig. 14. (A) Chromatogram of a serum sample from a patient suffering from severe depression. (B) Chromatogram of the same serum sample co-injected with inosine. (C) Purine nucleoside phosphorylase peak shift of patient serum sample. Conditions are the same as in Fig. 10 except as follows gradient linear from 1 to 40% B in 35 min. Reprinted with permission from Krstulovic et al. (K3I). Copyright by Elsevier Scientific Publishing Company, Amsterdam.

The most recent application of RPLC to the analysis of enzymes has been reported by Halfpenny and Brown (HI). An assay for purine nucleoside phosphorylase, a key mediator in the purine salvage pathway, has been developed and optimal conditions for the analysis determined. Figure 20 illustrates the simultaneous separation of the substrate, inosine, and products, uric acid and hypoxanthine. In another analysis. Halfpenny and Brown (H2) developed an assay for hypoxanthine-guanine phos-phoribosyltransferase. Deficiency of this enzyme has been associated with Lesch-Nyhan syndrome as well as primary gout. The activity of the enzyme is determined by measurement of the decrease of the substrate, hypoxanthine, and increase in the product, inosine-5 -monophosphoric acid. A major advantage of using HPLC for enzyme assays is that the simultaneous measurement of both substrate and product reduces the error due to interference from competing enzymes. 

In a commercially available assay, serum NTP catalyzes the hydrolysis of IMP to yield inosine, which is then converted to hypoxanthine by purine-nucleoside phosphorylase (EC 2.4.2.1). Hypoxanthine is oxidized to urate with xanthine oxidase (EC 1.2.3.2). Two moles of hydrogen peroxide are produced for each mole of hypoxanthine liberated and converted to uric acid. The formation rate of hydrogen peroxide is monitored by a spectrophotometer at 510nm by the oxidation of a chromogenic system. The effect of ALPs on IMP is inhibited by p-glycerophosphate. This material is substrate for ALP but not for NTP, and by forming substrate complexes with the former enzyme, it reduces the proportion of the total ALP activity that is directed to the hydrolysis of the NTP substrate, IMP. ... 

Catabolism of the nucleotides (Figure 24-3, B) begins with removal of their ribose-linked phosphate, a process catalyzed by purine 5 -nucleotidase. Removal of the ribose moiety of inosine and guanosine by the action of purine-nucleoside phosphorylase forms hypoxanthine and guanine, both of which are converted to xanthme. Xanthine is converted to uric acid through the action of xanthine oxidase. 

The phosphorylase can catalyze the formation of inosine or deoxyinosine, and of guanosine or deoxyguano-sine, but not adenosine or deoxyadenosine. However, the last two nucleosides can be converted to inosine and deoxyinosine by adenosine deaminase. The normal function of the phosphorylase appears to be the formation... 

Purine nucleosides are cleaved by the action of purine nucleoside phosphorylase with the liberation of ribose 1-phosphate (Kl, PI). The enzyme is apparently specific for purines. The material from erythrocytes catalyzes the phosphorolysis of purine but not pyrimidine nucleosides (T6.) Purine phosphorylase activity is found widespread in nature and in many animal tissues (FIO). Friedkin and Kalckar investigated an enzyme capable of cleaving purine deoxynucleosides to the aglycone and deoxy-ribose 1-phosphate. They concluded that the enzyme was identical to that which splits purine ribonucleosides (F8, F9). This enzyme is capable of degrading inosine, xanthosine, and guanosine to forms readily attacked by other enzymes. In so doing, it permits living cells to retain the ribose and deoxyribose moieties. 

Watanabe et al. (1986) developed a sequence sensor for the successive assay of hypoxanthine (HX) and inosine (HXR) by arranging nucleoside phosphorylase (EC 2.4.2.1) and xanthine oxidase (EC 1.2.3.2) in spatially separated layers in front of an oxygen probe. Nucleoside phosphorylase was... 

Gotoh et al. (1988) constructed a sensor for hypoxanthine by immobilizing xanthine oxidase on the gate of an amorphous silicon ISFET. The enzyme-FET responded to HX in the range 0.02-0.1 mmol/1. By coimmobilization of nucleoside phosphorylase, inosine could be measured in the same concentration range. 

Since inorganic phosphate is indispensable for the nucleoside phosphorylase reaction, the phosphate concentration can be converted into an oxygen signal by using a nucleoside phosphorylase-xanthine oxidase sequence electrode (Watanabe et al., 1987 Watanabe, 1988). 2 mmol/1 of inosine has been shown to be necessary for optimal sensitivity to phosphate ion. The measuring range was 0.1-1 mmol/1 and the sensor could be used for 70 assays. 

Figure 5.15 Equilibrium perturbation. Absorbance changes at 250 nm consequent upon the addition of E. coli purine nucleoside phosphorylase (ISOpg in 5 iL) to a solution (3mL) containing [l - H]inosine (1.99mM), hypo-xanthine (O.lOOmM), ribose-1-phosphate (O.SOmM) and inorganic phosphate (ll.SmM) in 0.2 M glycine-HCl buffer, pH 9.4, in a 1cm pathlength cuvette. ...

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