Metabolisms of Nucleosides in Bacteria

The mechanism of trans-N-ribosylation in Corynebacterium sepedonicum was investigated. Using the DEAE-cellulose colum chromatography, this enzyme activity was divided into two fractions. One cleaved uridine to uracil and ribose phosphate, and the other decomposed inosine into hypoxanthine and ribose phosphate, in the presence of inorganic phosphate. The ribose phosphate was isolated and crystallized.

Several analytical data indicated that the ribose phosphate was ribose-1-phosphate. These two enzyme fractions catalyzed the formation of nucleosides from ribose-1-phosphate and bases.

Most of bacteria, which had the activity to transfer N-ribosyl group between purine and pyrimidine, could synthesize the nucleoside from base and ribose-1-phosphate.     

Interrelations of NAD and adenosine transformation in the rat liver

The interrelationship of NAD and adenosine (inosine) conversions in the rat liver is investigated. The ratio of products of NAD+ conversions (ADP-ribose, inosine, hypoxanthine and ribose phosphates) are established. AMP and adenosine are not detected, which indicates an availability of different activities of the corresponding enzymes. It is shown that under conditions of the high inorganic phosphate concentration (33 mM) ribose-1-phosphate, formed in the purine nucleoside phosphorylase reaction, is accumulated due to the phosphoribomutase inhibition, but in the presence of NAD+ the utilization of ribose phosphate increases significantly. Nicotinamide inhibits the NAD+-glycohydrolase reaction in the system containing 33 mM phosphate, NAD+ and adenosine and simultaneously it lowers the utilization of ribose.     

The existence 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 Balb/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 nuceloside 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.     

Release of14C-adenine derivatives from cerebral cortical slices: Effect of phenytoin and phenobarbital

Both ouabain, 0.1 mM, and veratridine, 0.05 mM, increased the release of¹⁴C-labeled compounds from rat cortical slices prelabeled with¹⁴C-adenine and incubated in vitro. The increment in radioactivity released by both depolarizing agents was almost entirely a result of increases in adenosine, inosine, and hypoxanthine. However, the distribution of these three compounds in the ouabain-induced efflux (adenosine, 12%; inosine, 51%; hypoxanthine, 36%) contrasted with that evoked by veratridine (adenosine, 42%; inosine, 15%; hypoxanthine, 38%). Phenytoin significantly reduced the efflux of¹⁴C-labeled compounds produced by both ouabain and veratridine, but phenobarbital had no effect. The intracortical injection of adenosine, inosine, and hypoxanthine has been shown to induce epileptiform discharges in rats, and it is suggested that the inhibitory effect of phenytoin on the release of adenine derivatives may play a role in its antiepileptic action.     

Inosine metabolism in the rat

1. Uptake and subsequent metabolism of purine and ribose moieties was monitored after intravenous administration of doubly labelled inosine. 2. More than 95% was cleared from the plasma within 5 min, and 99% within 20 min. 3. Approx. 50% of the 160 mumol total was rapidly incorporated into liver and kidney. Kidney removed the greatest amount (21 mumol/g wet wt.), about 10-fold more than heart, lung or liver. Lung and heart accounted for only 3%. These tissues then lost radioactivity during the remainder of the experiment. Radioactivity in the skeletal muscle, in contrast, increased from 8% of the injected dose at 5 min to 40% at 60 min. 4. In liver, kidney, heart and lung there was a significant difference in the fate of inosine. After initial incorporation of inosine, kidney predominantly lost inosine; heart preferentially lost purines; lung preferentially lost ribose radioactivity; and in liver the ribose radioactivity was rapidly lost, whereas purine was retained. Some of the ribose moiety was metabolized to glucose, presumably in the liver, and then released into the blood. Ribose radioactivity (probably as glucose) and radioactive hypoxanthine accumulated in skeletal muscle throughout the experiment. 5. Inosine caused a rapid and prolonged increase in the blood glucose content, from 6 to 15 mM in 60 min. This was accompanied by a small increase in plasma insulin. 6. It is concluded that the purine and ribose radioactivity lost from the kidney, liver and other tissues becomes incorporated into skeletal muscle.     

Adenine nucleotide metabolism in Azotobacter vinelandii. Two metabolic pathways of AMP degradation

AMP-degrading pathways in Azotobacter vinelandii cells were investigated. AMP nucleosidase (EC 3.2.2.4) was rapidly synthesized and reached a maximum at 24 h, while the activity of 5-nucleotidase (EC 3.1.3.5) specific for AMP, which was negligible during the logarithmic phase of the growth, first appeared in 24 h-cultures, and reached a maximum after complete exhaustion of sucrose from the growth medium (70 h).Cell-free extracts of A. vinelandii of 48 h-cultures hydrolyzed AMP to ribose 5-phosphate and adenine in the presence of ATP, and adenine was deaminated to hypoxanthine. When ATP was excluded, AMP was dephosphorylated to adenosine, which was further metabolized to inosine, and finally to hypoxanthine. Hypoxanthine thus formed was reutilized for the salvage synthesis of IMP under the conditions where 5-phosphoribosyl 1-pyrophosphate was able to be supplied. These results suggest that the levels of ATP can determine the rate of AMP degradation by the AMP nucleosidase- and 5-nucleotidase-pathways. The role of ATP in the AMP degradation was discussed in relation to the regulatory properties of AMP nucleosidase, inosine nucleosidase (EC 3.2.2.2) and adenosine deaminase (EC 3.5.4.4).     

Effects of salicylic acid on post-ischaemic ventricular function and purine efflux in isolated mouse hearts

Abstract

Acetyl salicylic acid (aspirin) is one of the most widely used drugs in the world. Various plasma concentrations of aspirin and its predominant metabolite, salicylic acid, are required for its antiarthritic (1.5–2.5 mM), anti-inflammatory (0.5–5.0 mM) or antiplatelet (0.18–0.36 mM) actions. A recent study demonstrated the inhibitory effects of both aspirin and salicylic acid on oxidative phosphorylation and ATP synthesis in isolated rat cardiac mitochondria in a dose-dependent manner (0–10 mM concentration range). In this context, the present study was conducted to determine the effects of salicylic acid on inosine efflux (a potential biomarker of acute cardiac ischaemia) as well as cardiac contractile function in the isolated mouse heart following 20 min of zero-flow global ischaemia. Inosine efflux was found at significantly higher concentrations in ischaemic hearts perfused with Krebs buffer fortified with 1.0 mM salicylic acid compared with those without salicylic acid (12575±3319 vs. 1437±348 ng ml^?1 min^?1, mean±SEM, n=6 per group, p<0.01). These results indicate that 1.0 mM salicylic acid potentiates 8.8-fold ATP nucleotide purine catabolism into its metabolites (e.g. inosine, hypoxanthine). Salicylic acid (0.1 or 1.0 mM) did not appreciably inhibit purine nucleoside phosphorylase (the enzyme converts inosine to hypoxanthine) suggesting the augmented inosine efflux was due to the salicylic acid effect on upstream elements of cellular respiration. Whereas post-ischaemic cardiac function was further depressed by 1.0 mM salicylic acid, perfusion with 0.1 mM salicylic acid led to a remarkable functional improvement despite moderately increased inosine efflux (2.7-fold). We conclude that inosine is a sensitive biomarker for detecting cardiac ischaemia and salicylic acid-induced effects on cellular respiration. However, the inosine efflux level appears to be a poor predictor of the individual post-ischaemic cardiac functional recovery in this ex vivo model.     

Distinct mechanisms of hypoxanthine and inosine transport in membrane vesicles isolated from Chinese hamster ovary and Balb 3T3 cells

Both enzyme-mediated group translocation and facilitated diffusion have been proposed as mechanisms by which mammalian cells take up purine bases and nucleosides. We have investigated the mechanisms for hypoxanthine and inosine transport by using membrane vesicles from Chinese hamster ovary cells (CHO), Balb/c 3T3 and SV3T3 cells prepared by identical procedures. Uptake mechanisms were characterized by analyzing intravesicular contents, determining which substrates could exchange with the transport products, assaying for hypoxanthine phosphoribosyltransferase activity, and measuring the stimulation of uptake of hypoxanthine by phosphoribosyl pyrophosphate ($\text{P}\text{Rib}\text{-PP}$).We found that the uptake of hypoxanthine in Balb 3T3 vesicles was stimulated 3–4-fold by $\text{P}\text{Rib}\text{-PP}$. The intravesicular product was predominantly IMP. The hypoxanthine phosphoribosyltransferase activity copurified with the vesicle preparation. These results suggest the possible involvement of this enzyme in hypoxanthine uptake in 3T3 vesicles. In contrast to the 3T3 vesicles, CHO vesicles prepared under identical procedures did not retain hypoxanthine phosphoribosyltransferase activity and did not demonstrate $\text{P}\text{Rib}\text{-PP}$-stimulated hypoxanthine uptake. The intravesicular product of hypoxanthine uptake in CHO vesicles was hypoxanthine. These results and data from our kinetic and exchange studies indicated that CHO vesicles transport hypoxanthine via facilitated diffusion. An analogous situation was observed for inosine uptake; CHO vesicles accumulated inosine via a facilitated diffusion mechanism, while in the same experiments SV3T3 vesicles exhibited a purine nucleoside phosphorylase-dependent translocation of the ribose moiety of inosine.     

Enzymatic Synthesis of 5-Methyluridine from Adenosine and Thymine with High Efficiency

5-Methyluridine (5MU) was synthesized efficiently from adenosine, thymine, and phosphate by a combination of adenosine deaminase (ADA), purine nucleoside phosphorylase (PUNP), pyrimidine nucleoside phosphorylase (PYNP), and xanthine oxidase (XOD). Adenosine was converted into inosine first by ADA. 5MU and hypoxanthine were synthesized from inosine and thymine by PUNP and PYNP. The hypoxanthine formed was converted into urate via xanthine by XOD. After inosine was completely consumed, an equilibrium state, in which 5MU, thymine, ribose-1-phosphate, and phosphate were involved, was achieved. At the equilibrium state, the maximum yield of 5MU was obtained. The yield of 5MU was 74%, when the initial concentrations of adenosine, thymine, and phosphate were 5 mM each. On the other hand, in the absence of ADA or XOD the yield of 5MU was 1.8%. Several kinds of nucleosides were also synthesized with high yield by the same method.     

Purine nucleoside phosphorylase. Inosine hydrolysis, tight binding of the hypoxanthine intermediate, and third-the-sites reactivity.

Purine nucleoside phosphorylase from calf spleen is a trimer which catalyzes the hydrolysis of inosine to hypoxanthine and ribose in the absence of inorganic phosphate. The reaction occurs with a turnover number of 1.3 x 10(-4) s-1 per catalytic site. Hydrolysis of enzyme-bound inosine occurs at a rate of 2.0 x 10(-3) s-1 to form a stable enzyme-hypoxanthine complex and free ribose. The enzyme hydrolyzes guanosine; however, a tightly-bound guanine complex could not be isolated. The complex with hypoxanthine is stable to gel filtration but can be dissociated by acid, base, or mild denaturing agents. Following gel filtration, the E.hypoxanthine complex dissociates at a rate of 1.9 x 10(-6) s-1 at 4 degrees C and 1.3 x 10(-4) s-1 at 30 degrees C. The dissociation constant for the tightly-bound complex of enzyme-hypoxanthine is estimated to be 1.3 x 10(-12) M at 30 degrees C on the basis of the dissociation rate. The stoichiometry of the reaction is 1 mol of hypoxanthine bound per trimer. The reaction is reversible since the same complex can be formed from enzyme and hypoxanthine. Addition of ribose 1-phosphate to the complex results in the formation of inosine without release of hypoxanthine. Thus, the complex is catalytically competent. Inorganic phosphate or arsenate prevents formation of the tightly-bound E.hypoxanthine complex from inosine or hypoxanthine. Direct binding studies with hypoxanthine in the presence of phosphate result in 3 mol of hypoxanthine bound per trimer with a dissociation constant of 1.6 microM. In the absence of phosphate, three hypoxanthines are bound, but higher hypoxanthine concentrations cause the release of two of the hypoxanthines with an apparent inhibition constant of 130 microM. The results establish that enzymatic contacts with the nucleoside alone are sufficient to destabilize the N-glycosidic bond. In the absence of phosphate, water attacks slowly, causing net hydrolysis. The hydrolytic reaction leaves hypoxanthine stranded at the catalytic site, tightly bound to the enzyme with a conformation related to the transition state. In the phosphorolysis reaction, ribose 1-phosphate causes relaxation of this conformation and rapid release of hypoxanthine.     

Studies on the “Salvage” Synthesis of Ribonucleosides and Their 5′-Phosphates by Microorganisms

Studies on the “salvage” synthesis of ribonucleosides and their 5′-phosphates from nucleic acid bases by microorganisms were undertaken. After screening test of less than one hundred strains of type culture, it was found that inosine was produced from hypoxanthine by Arthrobacter ureafaciens, A. simplex, Flavobacterium aquatile and F. suaveolens.

In certain conditions, inosine was further oxidized and hydrolyzed into xanthosine, uric acid and etc.

As for the conditions of cultivation and reaction, the components of the medium and pH of the culture medium were important factors.

Using the standard method, the yield of inosine from hypoxanthine by F. suaveolens reached more than 60%, and the conversion was stoichiometric and any other by-products were not detected.

Inosine, xanthosine, guanosine and uridine were produced from adenine, xanthine, guanine and uracil, respectively, by F. suaveolens.     

Inosine synthesis and phosphoribosylpyrophosphate availability in rat liver cells in the presence of allopurinol

In the presence of allopurinol, apparent phosphoribosylpyrophosphate (PP-ribose-P) availability as measured by adenine incorporation into ribonucleotides was decreased in rat liver cells, hypoxanthine incorporation into ribonucleotides was increased, and there was a large synthesis of inosine from hypoxanthine. Inosine was formed directly by the reversal of the purine nucleoside phosphorylase reaction which was very rapid in liver cells. We tested the hypothesis that utilization of ribose 1-phosphate for inosine synthesis could decrease PP-ribose-P availability. Our results indicate that the apparent decrease of PP-ribose-P availability in the presence of allopurinol was due to competition between adenine and hypoxanthine salvage pathways into nucleotides, and not to the synthesis of inosine.     

Extracellular Cyclic AMP—Adenosine Pathway in Isolated Adipocytes and Adipose Tissue

Objective: Our goal was to evaluate the presence and lipolytic impact of the extracellular cyclic adenosine monophosphate (AMP)–adenosine pathway in adipose tissue. Research Methods and Procedures: Sixteen miniature Yucatan swine (Sus scrofa) were used for these in vitro and in situ experiments. Four microdialysis probes were implanted into subcutaneous adipose tissue and perfused at 2 μL/min with Ringer's solution containing no addition, varying levels of cyclic AMP, 10 μM isoproterenol, or 10 μM isoproterenol plus 1 mM α,β‐methylene adenosine 5′‐diphosphate (AMPCP), a 5′‐nucleotidase inhibitor. Dialysate was assayed for AMP, adenosine, inosine, hypoxanthine, and glycerol. Freshly isolated adipocytes were incubated with buffer, 1 μM isoproterenol, or 1 μM isoproterenol plus 0.1 mM AMPCP, and extracellular levels of AMP, adenosine, inosine, hypoxanthine, and glycerol were measured. Results: Perfusion of adipose tissue with exogenous cyclic AMP caused a significant increase in AMP and adenosine appearance. Perfusion with AMPCP, in the presence or absence of isoproterenol, significantly increased the levels of AMP and glycerol, whereas it significantly reduced the level of adenosine and its metabolites. However, the AMPCP‐provoked increase in lipolysis observed in situ and in vitro was not temporally associated with a decrease in adenosine. Discussion: These data suggest the existence of a cyclic AMP—adenosine pathway in adipocytes and adipose tissue. The role of this pathway in the regulation of lipolysis remains to be clarified.     

Utilization of exogenous purine compounds in Bacillus cereus. Translocation of the ribose moiety of inosine.

Intact cells of Bacillus cereus catalyze the breakdown of exogenous AMP to hypoxanthine and ribose 1-phosphate through the successive action of 5'-nucleotidase, adenosine deaminase, and inosine phosphorylase. Inosine hydrolase was not detectable, even in crude extracts. Inosine phosphorylase causes a "translocation" of the ribose moiety (as ribose 1-phosphate) inside the cell, while hypoxanthine remains external. Even though the equilibrium of the phosphorolytic reaction favors nucleoside synthesis, exogenous inosine (as well as adenosine and AMP) is almost quantitatively transformed into external hypoxanthine, since ribose 1-phosphate is readily metabolized inside the cell. Most likely, the translocated ribose 1-phosphate enters the sugar phosphate shunt, via its prior conversion into ribose 5-phosphate, thus supplying the energy required for the subsequent uptake of hypoxanthine in B. cereus.     

Hypoxanthine transport through the blood-brain barrier

The unidirectional influx of hypoxanthine across cerebral capillaries, the anatomical locus of the blood=brain barrier, was measured with an in situ rat brain perfusion technique employing [³H]hypoxanthine. Hypoxanthine was transported across the blood-brain barrier by a saturable system with a one-half saturation concentration of approximately 0.4 mM. The permeability-surface area product was 3×10^–4 sec^–1 with a hypoxanthine concentration of 0.02 M in the perfusate. Adenine (4 mM) and uracil and theophylline (both 10 mM), but not inosine (10 mM) or leucine (1 mM), inhibited hypoxanthine transfer through the blood-brain barrier. Thus, hypoxanthine is transported through the blood-brain barrier by a high-capacity, saturable transport system with a half-saturation concentration about 100 times the plasma hypoxanthine concentration. Although involved in the transport hypoxanthine from blood into brain, this system is not powerful enough to transfer important quantities of hypoxanthine from blood into brain.     

A general method of alpha-aminoaldehyde synthesis using alcohol dehydrogenase

A method for measuring ribose 1-phosphate in cell extracts is described. Cell extracts are first fractionated on polyethyleneimine-impregnated cellulose columns to remove nucleoside and base components which otherwise interfere with the enzymatic assay. Ribose 1-phosphate in the eluate is made limiting for the conversion of [¹⁴C]hypoxanthine to [¹⁴C]inosine in the presence of purine nucleoside phosphorylase. Labeled substrate and product are then easily separated on boronate gel columns or by paper chromatography.     

Conversion of Hypoxanthine Derivatives to Guanine Derivatives by Bacillus subtilis

By successive mutagenic treatments including transduction with bacteriophage SP–10, ultraviolet light irradiation and N-methyl-N′-nitro-N-nitrosoguanidine treatments, a mutant, strain No. 322, capable of converting exogenously supplemented hypoxanthine or inosine to guanine and guanosine, was derived from an adenine-less, IMP-producing mutant of Bacillus subtilis IAM 1145. Strain No. 322 was an adenine-leaky mutant lacking GMP-reductase, adenase, and 5′-nucleotidase. The strain effectively accumulated guanine and guanosine in the culture fluid, when grown in the presence of hypoxanthine or inosine, while it failed to convert exogenously supplemented IMP to the guanine derivatives.     

De novo engineering and metabolic flux analysis of inosine biosynthesis in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Wild-type B. subtilis strain W168 was de novo engineered for inosine biosynthesis. Inactivation of deoD and purA led to 0.15 ± 0.04 and 6.44 ± 0.39 g inosine/l yields, respectively. The deoD purA double mutant accumulated 7.6 ± 0.34 g inosine/l, with a 4.7% (w/w) conversion ratio from glucose to inosine. Comparative metabolic flux analysis revealed that the fluxes from inosine to hypoxanthine and from inosine monophosphate to adenosine monophosphate in the double mutant decreased to 14.0 and 0.61% of those in the wild-type strain. The major role of purA was demonstrated when inactivation of deoD and purA were found to contribute additively to inosine accumulation. This work is expected to contribute to the improvement of the fermentative production of purine nucleosides in the microbial industry.     

Substitution of inosine for yeastolate in the culture medium forDrosophila cells

Summary The nutritional requirement ofDrosophila cells (GM₁ and GM₂) was studied. TC Yeastolate contained in the medium forDrosophila cell culture was found to be replaceable with adenosine or inosine without appreciable changes in the generation time of cells. The optimal concentration of either adenosine or inosine was 0.01 mM. Whereas adenosine manifested cell toxicity at concentrations higher than 0.1 mM, in the case of inosine, such an inhibitory effect was not observed up to and at the concentration of 1.0 mM. Further-more, the plating efficiency at cell densities as low as 2×10³ cells per cm² was raised from 0 to 10% by supplementing inosine (0.1 mM) for the TC Yeastolate. Therefore inosine is in practice more useful than adenosine. Experiments using radioactive nucleosides suggested that both adenosine and inosine were exclusively incorporated into RNA as adenosine-monophosphate.     

Ribose 1-phosphate and inosine activate uracil salvage in rat brain

The purpose of this study was to determine the mechanism by which inosine activates pyrimidine salvage in CNS. The levels of cerebral inosine, hypoxanthine, uridine, uracil, ribose 1-phosphate and inorganic phosphate were determined, to evaluate the Gibbs free energy changes (deltaG) of the reactions catalyzed by purine nucleoside phosphorylase and uridine phosphorylase, respectively. A deltaG value of 0.59 kcal/mol for the combined reaction inosine+uracil <==> uridine+hypoxanthine was obtained, suggesting that at least in anoxic brain the system may readily respond to metabolite fluctuations. If purine nucleoside phosphorolysis and uridine phosphorolysis are coupled to uridine phosphorylation, catalyzed by uridine kinase, whose activity is relatively high in brain, the three enzyme activities will constitute a pyrimidine salvage pathway in which ribose 1-phosphate plays a pivotal role. CTP, presumably the last product of the pathway, and, to a lesser extent, UTP, exert inhibition on rat brain uridine nucleotides salvage synthesis, most likely at the level of the kinase reaction. On the contrary ATP and GTP are specific phosphate donors.