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Nucleotide mutations in purA gene and pur operon promoter discovered in guanosine- and inosine-producing Bacillus subtilis strains.

The promoter region of the pur operon, which contains 12 genes for inosine monophosphate biosynthesis from phosphoribosylpyrophosphate, and the purA gene, encoding the adenylosuccinate synthetase, were compared among wild-type and three purine-producing Bacillus subtilis strains. A single nucleotide deletion at position 55 (relative to translation start site) in purA gene was found in a high inosine-producing strain and in a high guanosine-producing strain, which correlates with the absence of adenylosuccinate synthetase activity in these strains. Within the pur operon promoter of high guanosine-producing strain, in addition to a single nucleotide deletion in PurBox1 and a single nucleotide substitution in PurBox2, there were 4 substitutions in the flanking region of the PurBoxes and 32 nucleotide mutations in the 5' untranslated region. These mutations may explain the purine accumulation in purine-producing strains and be helpful to the rational design of high-yield recombinant strains.

Adenylosuccinate Synthase↗

Assay method for monitoring the inhibitory effects of antimetabolites on the activity of inosinate dehydrogenase in intact human CEM lymphocytes.

A rapid and convenient method has been developed to monitor the inhibition of inosinate (IMP) dehydrogenase by antimetabolites in intact human CEM lymphocytes. This method is based on the determination of 3H release from [2,8-3H]hypoxanthine ([2,8-3H]Hx) or [2,8-3H]inosine ([2,8-3H]Ino). The validity of this procedure was assessed by evaluating IMP dehydrogenase inhibition in intact CEM cells by the well-known IMP dehydrogenase inhibitors ribavirin, mycophenolic acid and tiazofurin. As reference materials, several compounds that are targeted at other enzymes in de novo purine nucleotide anabolism (i.e. hadacidine, acivicin) or catabolism (i.e. 8-aminoguanosine, allopurinol) were evaluated. There was a strong correlation between the inhibitory effects of the IMP dehydrogenase inhibitors (ribavirin, mycophenolic acid, tiazofurin) on 3H release from [2,8-3H]Hx and [2,8-3H]Ino in intact CEM cells and their ability to decrease intracellular GTP pool levels. The other compounds (hadacidine, acivicin, 8-aminoguanosine, allopurinol) had no marked effect on 3H release from [2,8-3H]Hx. Using this method, we demonstrated that the novel ribavirin analogue, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide, is a potent inhibitor of IMP dehydrogenase in intact cells.

Adenosine Triphosphate↗

Adverse drug reactions to azathioprine therapy are associated with polymorphism in the gene encoding inosine triphosphate pyrophosphatase (ITPase).

Adverse drug reactions to azathioprine (AZA), the pro-drug of 6-mercaptopurine (6-MP), occur in 15% to 28% of patients and the majority are not explained by thiopurine methyltransferase (TPMT) deficiency. Inosine triphosphate pyrophosphatase (ITPase) deficiency results in the benign accumulation of the inosine nucleotide ITP. 6-MP is activated through a 6-thio-IMP intermediate and, in ITPase deficient patients, potentially toxic 6-thio-ITP is predicted to accumulate. The association between polymorphism in the ITPA gene and adverse drug reactions to AZA therapy was studied in patients treated for inflammatory bowel disease. Sixty-two patients with inflammatory bowel disease suffering adverse drug reactions to AZA therapy were genotyped for ITPA 94C>A and IVS2 + 21A>C polymorphisms, and TPMT*3A, *3C, *2 polymorphisms. Genotype frequencies were compared to a consecutive series of 68 controls treated with AZA for a minimum of 3 months without adverse effect. The ITPA 94C>A deficiency-associated allele was significantly associated with adverse drug reactions [odds ratio (OR) 4.2, 95% confidence interval (CI) 1.6-11.5, P = 0.0034]. Significant associations were found for flu-like symptoms (OR 4.7, 95% CI 1.2-18.1, P = 0.0308), rash (OR 10.3, 95% CI 4.7-62.9, P = 0.0213) and pancreatitis (OR 6.2,CI 1.1-32.6, P = 0.0485). Overall, heterozygous TPMT genotypes did not predict adverse drug reactions but were significantly associated with a subgroup of patients experiencing nausea and vomiting as the predominant adverse reaction to AZA therapy (OR 5.5, 95% CI 1.4-21.3, P = 0.0206). Polymorphism in the ITPA gene predicts AZA intolerance. Alternative immunosuppressive drugs, particularly 6-thioguanine, should be considered for AZA-intolerant patients with ITPase deficiency.

Adolescent↗

Affinity chromatography on immobilised nucleotides. The synthesis, specificity and applications of immobilised inosine 5'-monophosphate.

The synthesis and characterisation of two IMP analogues, 8-(6-aminohexyl)-ionosine 5'-monophosphate, Ahx8IMP, and inosine 2',3'-O-[1-(6-aminohexyl)-levulinic acid amide]-acetyl 5'-monophosphate, (AhxLvn)2',3'IMP, is described. These analogues were attached to CNBr-activated agarose through the terminal amino group of the spacer molecule. The immobilised-IMP analogues displayed specificity for the inosine-nucleotide-dependent enzyme, IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.2.1.14) but not for the NAD+-dependent enzymes, L-alanine and L-acetate dehydrogenases. Escherichia coli IMP dehydrogenase could be eluted biospecifically from immobilised 8-substituted and ribose-substituted IMP adsorbents with IMP, XMP and GMP. Multiple peaks of enzyme activity in the elution profiles were interpreted in terms of aggregation of the enzyme. A protocol for the large-scale purification of E. coli IMP dehydrogenase is proposed. Homogeneous enzyme of specific activity 9.1 units/mg was obtained in 50% overall yield, representing 14 mg pure protein from a 20-1 culture of E. coli. The two IMP analogues were inactive as substrates in the IMP dehydrogenase reaction.

Animals↗

Comparison of the self-association properties of the 5'-triphosphates of inosine (ITP), guanosine (GTP), and adenosine (ATP). Further evidence for ionic interactions in the highly stable dimeric [H2(ATP)]2(4-) stack.

The concentration dependence of the chemical shifts for the hydrogens H-2, H-8 and H-1' of ITP and for H-8 and H-1' of GTP has been measured in D2O at 25 degrees C under several degrees of protonation in the pD range 1.2-8.4. For reasons of comparison, inosine and guanosine have been included in the study The results are consistent with the isodesmic model of indefinite noncooperative stacking. The association constants for the nucleosides (Ns) inosine and guanosine decrease with increasing protonation: Ns greater than D(Ns)+/Ns in a 1:1 ratio greater than D(Ns)+. In contrast, a maximum is observed with ITP and GTP; the stacking tendency of GTP following the series: GTP4- less than or equal to D(GTP)3- (K approximately 0.7 M-1) less than D(GTP)3-/D2(GTP)2- in a 1:1 ratio (K approximately 2.9 M-1) greater than D2(GTP)2- greater than D3(GTP)- (K approximately 1.5 M-1). The order of the series with ITP corresponds to that with GTP, but the association constants are slightly smaller. At the maximum of the self-association tendency the triphosphate residue has only a minor influence; this follows from the fact that the association constants for the 1:1 ratios of Ino/D(Ino)+ and D(ITP)3-/D2(ITP)2- are identical within experimental error; this holds also for Guo/D(Guo)+ and D(GTP)3-/D2(GTP)2-; in all these pairs the K-7 site is 50% protonated. Comparison of the association constant for the deprotonated species shows that here charge effects, i.e. repulsion between the negatively charged triphosphate chains, are important: Ino (K approximately 3.3 M-1) greater than ITP4- (K approximately 0.4 M-1) and Guo (K approximately 8 M-1) greater than GTP4- (K approximately 0.8 M-1). In addition the series holds: Ado (K approximately 15 M-1) greater than Guo greater than Ino. However, most important is the comparison of the ITP and GTP series with previous data for ATP: ATP4- (K approximately 1.3 M-1) less than D(ATP)3- (2.1 M-1) less than 1:1 ratio of D(ATP)3-/D2(ATP)2- (6 M-1) much less than D2(ATP)2- (approximately 200 M-1) much greater than D3(ATP)- (K less than or equal to 17 M-1).(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

Suppression of frequently recurring genital herpes: acyclovir v inosine pranobex.

The suppressive action of acyclovir and inosine pranobex was compared in a randomised double blind controlled trial in patients with frequently recurring genital herpes. Fourteen patients received acyclovir and 17 inosine pranobex. Treatment continued for 12 weeks. The time to the first recurrence was significantly longer and the frequency of recurrences significantly less in the recipients of acyclovir. No important side effects were noted. It is concluded that acyclovir is the treatment of choice to suppress often recurring genital herpes.

Acyclovir↗

Guanosine triphosphate catabolism in human and rabbit erythrocytes: role of reductive deamination of guanylate to inosinate.

The reductive deamination of guanylate to inosinate was demonstrable but occurred at low rates in human and rabbit erythrocytes incubated in vitro with or without glucose. However, the process was considerably accelerated in erythrocytes incubated with deoxyglucose. In human erythrocytes incubated with deoxyglucose, deamination was the major pathway of catabolism of guanylate; little or no guanylate was dephosphorylated. In rabbit erythrocytes, guanylate was both deaminated and dephosphorylated. Inosinate formed from guanylate was metabolized only by dephosphorylation in human erythrocytes, but in rabbit erythrocytes, it was also converted to xanthylate.

Animals↗

Study of the antiallergic component of inosine pranobex (Inosiplex, Isoprinosine).

Inosine pranobex (INPX) at concentrations greater than 10(-3) M inhibits significantly the concanavalin A or antihuman IgE induced histamine release from human mast cells. The inosine moiety of the compound does not contribute to this effect, but rather interferes with it. The other component, the salt of dimethylaminopropanol and acetamidobenzoic acid, produces a small but significant inhibition of the histamine release already at 10(-6) M. This effect is due to the acetamidobenzoic acid although the base (dimethylaminopropanol) adds to it. With increasing incubation time, the salt of dimethylaminopropanol and acetamidobenzoic acid looses its histamine release inhibiting effect.

Concanavalin A↗

The effect of inosine, pyruvate, and inorganic phosphate on 2,3-diphosphoglycerate, adenine, and hypoxanthine nucleotide synthesis in outdated human erythrocytes.

Phosphate compounds of red cells stored at 4 degrees C. in acid-citrate-dextrose solution for 15 to 44 days, incubated at 37 degrees C. for 2 and 4 hours with inosine, pyruvate, and inorganic phosphate, and then stored for 2 weeks have been examined by ion-exchange column chromatography. After 2 and 4 hours of incubation the 2,3-diphospholycerate level increased to 1,385 and 1,819 mumoles per 100 ml. of erythroeytes, respectively. After 4 hours of incubation the content of AIP increased to about 80 per cent of the physiologic level, while the content of AMP and ADP decreased considerably. These data suggest that the elevation of ATP is in part connected with the phosphorylation of AMP and ADP. The incorporation of inosine or hypoxanthine into the IMP and ITP has been proved. Four hours incubation period caused the synthesis of IMP and ITP to the mean value of 187 and 141 mumoles per 100 ml. of erythrocytes, respectively. No evidence of IDP formation was observed during the chromatographic separations of erythrocytic phosphate compounds. Storage of the erythrocyte suspension which has been incubated for 4 hours leads to the slower decomposition of 2,3-diphosphoglycerate, ATP, ITP, and IMP, and to the increase of AMP and ADP.

Adenine Nucleotides↗

Inactivation of the bovine heart mitochondrial F1-ATPase by 5'-p-fluorosulfonylbenzoyl[3H]inosine is accompanied by modification of tyrosine 345 in a single beta subunit.

The inactivation of the bovine heart mitochondrial F1-ATPase by 5'-p-fluorosulfonylbenzoylinosine (FSBI) proceeds with pseudo-first order kinetics. The rate of inactivation increased from pH 7 to 9 revealing a pKa of about 8.2. When a tryptic digest of the enzyme which had been inactivated with 5'-p-fluorosulfonylbenzoyl[3H]inosine ([3H]FSBI) was submitted to reversed phase high pressure liquid chromatography, a single major peak of radioactivity, T1, was resolved. Amino acid sequence analysis of purified peptide fragments derived from T1 showed that the modification of beta-Tyr-345 is responsible for inactivation of the enzyme. Complete inactivation of the enzyme by [3H]FSBI is estimated to proceed with modification of 0.8 mol of beta-Tyr-345/mol of enzyme. Another notable observation is that inosine triphosphatase (ITPase) activity catalyzed by F1 from bovine heart mitochondria is much more sensitive to inactivation by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) than is ATPase activity. Whereas complete inactivation of ATPase activity by FSBA has been shown to proceed with the mutually exclusive modification of Tyr-368 or His-427 in all three copies of the beta subunit (Bullough, D. A., and Allison, W. S. (1986) J. Biol. Chem. 261, 5722-5730), it is shown here that complete inactivation of ITPase activity by FSBA is accompanied by modification of these residues in only one copy of the beta subunit. Inactivation of both the ATPase and ITPase activities of the enzyme by FSBI proceeds with modification of Tyr-345 in a single copy of the beta subunit.

Adenosine↗

[Pathways of inosine monophosphate transformation in the chicken liver].

The rates of distribution of the radioactive precursor label de novo and of the reutilization pathways of inosine monophosphate biosynthesis between adenine and guanine nucleotides in the acid-soluble fraction and RNA from chicken liver were determined. The activity of enzymes involved in inosine monophosphate conversion into adenine or guanine nucleotides or in uric acid biosynthesis was determined. It was shown that in chicken liver the activity of IMP-dehydrogenase and the rate constant for the label ([14C]glycine) incorporation into the guanine nucleotide pool is 3.5 times as high as that of adenyl succinate synthetase and the rate constant for the label liberation into the adenine nucleotide pool. The rate of xanthine synthesis is by one order of magnitude more than that of hypoxanthine and uric acid. The rate of IMP synthesis from [14C]hypoxanthine via the guanine nucleotide pathway is also higher in this case. Hence, in chicken liver IMP oxidation to XMP seems to proceed with a high efficiency and can thus be used, in addition to dephosphorylation, for ammonia extrusion.

Adenine Nucleotides↗

Inosine analogs. Their metabolism in mouse L cells and in Leishmania donovani.

The growth of Leishmania donovani promastigotes and mouse L cells is differentially inhibited by several inosine analogs with modifications in the imidazole ring. The protozoal and mammalian cells also demonstrate differential metabolism of these analogs. 7-Deazainosine, 7-thia-7,9-dideazainosine, and formycin B were converted to their respective ATP analogs by both cell types. 8-Azainosine was converted to a GTP analog by mouse L cells; L. donovani did not metabolize this nucleoside. 9-Deazainosine and allopurinol riboside were metabolized only to their respective IMP analogs by L cells. L. donovani metabolized 9-deazainosine and allopurinol riboside to their ATP analogs and also metabolized 9-deazainosine to its GTP analog. All nucleosides studied were resistant to cleavage by either organism. From metabolism studies in the presence of a specific enzyme inhibitor, it was deduced that allopurinol riboside, formycin B, and 9-deazainosine were phosphorylated by at least two different routes in the mouse L cells. The metabolism of formycin B was inhibited 65% by the adenosine kinase inhibitor, 5-iodotubercidin, whereas the metabolism of allopurinol riboside (14% inhibition) and 9-deazainosine (0% inhibition) was only slightly affected by this inhibitor. The metabolism of allopurinol riboside and 9-deazainosine by L. donovani was not affected by 5-iodotubercidin. In contrast to the results of L cells, the metabolism of formycin B by L. donovani was also not affected by 5-iodotubercidin. The abilities of mouse L cells and L. donovani to metabolize these inosine analogs to the corresponding nucleotide analogs of ATP or GTP may be considered to be an activating step and correlates well with the respective cytotoxic effects of these compounds.

Adenosine Kinase↗

The interaction of metal ions with nucleic acids. A nuclear magnetic resonance relaxation time study of the copper(II)-inosine 5'-monophosphate system in solution.

The nature of binding between copper ions and inosine 5'-monophosphate was studied using 13C, 1H, and 31P nuclear magnetic resonance techniques. The results imply that at least two species are present in the solution with the metal to ligand ratio of 1:1 and 1:2 (termed, respectively, ML and ML2); however, the ML complex predominates in the Cu(II)-IMP system. The results indicate that the copper ions bind directly to two different sites on the inosine ring i.e., to N7 and to N1. The distribution of the species MLRP7 and MLRP1 is nearly equal. The relative dipolar and scalar contributions to the proton and carbon-13 transverse relaxation have been assessed.

Copper↗

The existance of a group translocation transport mechanism in animal cells: uptake of the ribose moiety of inosine.

After exposure to inosine, transport-competent plasma membrane vesicles isolated from SV-40-transformed Bal/c 3T3 cells accumulate intravesicular ribose 1-PO4 at a concentration 200-fold greater than the extravesicular concentration. An analysis of the purine nucleoside phosphorylase activity distribution in various subcellular fractions, relative to other enzyme activities, indicated the presence of plasma membrane-associated purine nucleoside phosphorylase activity. The plasma membrane vesicles appear relatively impermeable to hypoxanthine. However, hypoxanthine, which is a competitive inhibitor of the transport reaction, is the only compound tested capable of mediating efflux of already accumulated ribose 1-PO4. In addition, hypoxanthine does not result in the efflux of transported uridine which is accumulated in these membrane vesicles as uridine. Exogenous ribose 1-PO4 neither results in counterflow nor does it inhibit the original uptake reaction. The following transport reaction is proposed: uptake occurs by group translocation, mediated by membrane-localized purine nucleoside phosphorylase. The data are consistent with sites for inosine and hypoxanthine being on the outer membrane surface whereas the ribose 1-PO4 site is only on the inner surface.

Adenine↗

Monovalent cation activation in Escherichia coli inosine 5'-monophosphate dehydrogenase.

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate with the concomitant reduction of NAD to NADH. Escherichia coli IMPDH is activated by K(+), Rb(+), NH(+)(4), and Cs(+). K(+) activation is inhibited by Li(+), Na(+), Ca(2+), and Mg(2+). This inhibition is competitive versus K(+) at high K(+) concentrations, noncompetitive versus IMP, and competitive versus NAD. Thus monovalent cation activation is linked to the NAD site. K(+) increases the rate constant for the pre-steady-state burst of NADH production, possibly by increasing the affinity of NAD. Three mutant IMPDHs have been identified which increase the value of K(m) for K(+): Asp13Ala, Asp50Ala, and Glu469Ala. In contrast to wild type, both Asp13Ala and Glu469Ala are activated by all cations tested. Thus these mutations eliminate cation selectivity. Both Asp13 and Glu469 appear to interact with the K(+) binding site identified in Chinese hamster IMPDH. Like wild-type IMPDH, K(+) activation of Asp50Ala is inhibited by Li(+), Na(+), Ca(2+), and Mg(2+). However, this inhibition is noncompetitive with respect to K(+) and competitive with respect to both IMP and NAD. Asp50 interacts with residues that form a rigid wall in the IMP site; disruption of this wall would be expected to decrease IMP binding, and the defect could propagate to the proposed K(+) site. Alternatively, this mutation could uncover a second monovalent cation binding site.

Binding Sites↗

Simultaneous determination of adenosine, inosine, hypoxanthine, xanthine, and uric acid in microdialysis samples using microbore column high-performance liquid chromatography with a diode array detector.

In the myocardial interstitial space, adenosine and its metabolites are important markers of ischemia, regulators of blood flow, and may produce cardioprotection against ischemia. A fast and sensitive method to assess the concentrations of adenosine and its metabolites is necessary to determine their involvement in mediating these effects. A method for the simultaneous determination of adenosine, inosine, hypoxanthine, xanthine, and uric acid in the interstitial fluid of the canine myocardium was developed using microdialysis, microbore column high-performance liquid chromatography, and a photo diode array detector (DAD). The microdialysis samples were injected directly onto a microbore C18 reverse-phase column without any prior sample preparation. Use of a DAD in this method provided many advantages. First, a DAD allowed the simultaneous detection of UV absorbance at multiple wavelengths, allowing the detection of each compound at their maximal UV absorbance. Further, the full UV absorption spectrum was recorded for each detected peak, confirming peak purity and identity. Using a microbore HPLC column and detection of UV absorbance at the maximal absorbance for each compound improve the sensitivity for all compounds. The detection limit of these compounds is 50 fmol (signal-to-noise ratio, S/N = 3). This method is useful in analyzing the temporal effect of a prolonged period of myocardial ischemia and reperfusion upon interstitial adenosine, inosine, hypoxanthine, xanthine, and uric acid concentrations in an in vivo canine model.

Adenosine↗

Inosine(15.1) hammerhead ribozymes for targeting the transthyretin-30 mutation.

The most common cause of hereditary amyloidosis (HA) is the val30met mutation in the transthyretin protein (TTR-met30). The mutation is caused by a mononucleic substitution from G to A (GUC to AUC) in the transthyretin gene resulting in the exchange for the amino acids valine to methionine in the corresponding protein sequence. The aim of our work was the development of a specific cleavage of TTR-30 mRNA using hammerhead ribozymes. We chemically modified nuclease stable hammerhead ribozymes to target the TTR-30 mRNA with high specificity. The exchange of adenosine(15.1) with inosine(15.1) in the catalytic core of the hammerhead ribozyme resulted in a change of the cleavable target sequence from N(16.2)U(16.1)H(17) to N(16. 2)C(16.1)H(17) without loss in ribozymal activity (Nucleic Acids Res. 26, 2279-2285, 1998). This modification allowed a specific cleavage of the TTR-30 mutation ("gCC Gug" to "gCC Aug"). In vitro experiments with TTR-30 mRNA demonstrated that the RNase stable inosine(15.1) hammerhead ribozyme cleaved the TTR-30 mRNA with 100% specificity and with a velocity of 0.23 min(-1), whereas no cleavage occured in the wildtype mRNA of TTR. In conclusion, the development of this NCH specific hammerhead ribozyme represents a promising tool for future in vivo therapeutic application for TTR-met30 induced hereditary amyloidosis.

Hydrolysis↗