Preservation of red blood cells with purines and nucleosides. II. Uptake and utilization of purines and nucleosides by stored red blood cells

The uptake of adenine, guanine, guanosine and inosine by stored red cells was investigated in whole blood and red cell resuspensions at initial concentrations of 0.25, 0.5 and 0.75 mM for adenine and 0.5 mM for the other additives using a rapid ion-exchange chromatographic microanalysis of purines and nucleosides in plasma and whole blood. Increasing adenine concentrations from 0.25 to 0.75 mM in blood elevated the adenine uptake from 0.3 up to 0.8 mmol/l red cells during 2 hours after collecting blood. The intra-/extracellular distribution ratio changed from 1 : 1.3 to 1: 1.7. Some 2 hours after withdrawing blood into CPD--solution with purines and nucleosides the uptake of adenine and guanine resulted in 40 per cent and 70 per cent respectively and of guanosine and inosine in 80 and 90 per cent respectively. The replacement of plasma by a resuspending solution gave the same uptake rates for purines and nucleosides. The nucleosides were rapidly split to purines and R-1-P and disappeared from blood during one week. Adenine and guanine were utilized to 80 to 90 per cent only after 3 weeks. During the same period the utilization of guanine was smaller by 40 per cent than that of adenine due to the different activity of the purine nucleoside phosphorylase for these substrates. The plasma of all analyzed blood samples contained hypoxanthine and inosine, but guanine and guanosine were detected only in those samples to which one of them was added. After 3 weeks of storage the highest concentration of hypoxanthine was found in CPD-AI blood with 600 microM in plasma and the highest concentration of synthesized inosine in CPD-AG blood with a concentration of 100 microM in plasma. Three ways of utilization of purines by stored red cells were discussed : the synthesis of nucleotide monophosphates, the formation of nucleosides, and the deamination. The portions of these ways change during storage. The most effective concentrations of adenine and guanosine in stored blood seems to be 0.25 and 0.5 mM respectively. The full utilization of the nucleoside requires the addition of inorganic phosphate.     

Sugar-free growth of mammalian cells on some ribonucleosides but not on others

It was shown earlier that a variety of vertebrate cells could grow indefinitely in sugar-free medium supplemented with either uridine or cytidine at greater than or equal to 1 mM. In contrast, most purine nucleosides do not support sugar-free growth for one of the following reasons. The generation of ribose-1-P from nucleoside phosphorylase activity is necessary to provide all essential functions of sugar metabolism. Some nucleosides, e.g. xanthosine, did not support growth because they are poor substrates for this enzyme. De novo pyrimidine synthesis was inhibited greater than 80% by adenosine or high concentrations of inosine, e.g. 10 mM, which prevented growth on these nucleosides; in contrast, pyrimidine synthesis was inhibited only marginally on 1 mM inosine or guanosine, but normal growth was only seen on 1 mM inosine, not on guanosine. The inhibition of de novo adenine nucleotide synthesis prevented growth on guanosine, since guanine nucleotides could not be converted to adenine nucleotides. Guanine nucleotides were necessary for this inhibition of purine synthesis, since a mutant blocked in their synthesis grew normally on guanosine. De novo purine synthesis was severely inhibited by adenosine, inosine, or guanosine, but in contrast to guanosine, adenosine and inosine could provide all purine requirements by direct nucleotide conversions.     

Effect of Adenosine and Deoxyadenosine on the Incorporation and Breakdown of Thymidine in Escherichia coli K-12

Deoxyadenosine (AdR) and adenosine (AR) enhance the incorporation of thymidine (TdR) into bacterial deoxyribonucleic acid (DNA) by the inhibition of TdR phosphorolysis in vivo. Neither of the purine nucleosides has an effect on the reaction catalyzed by TdR phosphorylase in vitro. AdR induces TdR phosphorylase and both purine nucleosides induce purine nucleoside phosphorylase. AR can stimulate uptake of more TdR into bacterial DNA than AdR.     

Adenine uptake in Neurospora crassa mycelium]

The uptake of 8-C14-adenine in N. crassa strain Lindegren (+) was studied. The ability of N. crassa cells to uptake adenine from the medium reaches maximum at the very beginning of the logarithmic stage of growth. Adenine enters the mycelium against the concentration gradient. The uptake of adenine is maximal at 25-30 degrees C, pH 4,6-4,8, and adenine concentration in the medium about 2-15X10(-6) M. The entry of adenine into the cells follows normal Michaelis-Menten kinetics, the apparent Km=0.83+/-0.02 micron. The uptake is inhibited at higher concentrations (10(-3)-10(-4) M) of adenine. 2,6-Diaminopurine, hypoxanthine, guanine, 8-azaadenine and 8-azaguanine inhibit the transport of adenine into the cell. Xanthine and cytosine do not affect the uptake of adenine. Adenine taken up into the cell is rapidly metabolized to AMP, ADP and ATP.     

Escherichia coli K-12 mutants capable of catabolizing purine nucleosides in the absence of purine nucleoside phosphorylase]

Strains of Escherichia coli K-12 defective in purine nucleoside phosphorylase (pup gene) formed on the medium with inosine as the source of carbon and energy phenotypical reversions for the ability of utilizing inosine as source of carbon or purines. The phenotypical suppression of the purine nucleoside phosphorylase deficiency is the result of the mutations (called pnd), which are mapped on the chromosome of E. coli beyond the region of the structural pup-gene location and have phenotypic manifestation distinct from that of pup+ allele: a) pnd mutants divide into some groups for the ability of utilizing several purine nucleosides, including xantosine that cannot be metabolized by pnd+ strains of E. coli; b) pnd mutations do not restore the ability of purine auxotrophs (pur) defective in purine nucleoside phosphorylase (pup) and adenine phosphoribosyltransferase (apt) to grow on the medium with adenine as the sole source of purines. Cell-free extracts of pnd mutants fail to degrade the guanine nucleosides in the absence of phosphate or arsenate ions. These data (and also the ability of pnd mutants to utilize both purine ribonucleosides and deoxyribonucleosides) seem to indicate that the activities induced by pnd mutations are phosphorylase activities.     

Purine metabolism in microplasmodia of Physarum polycephalum.

The uptake and utilization of purine nucleosides and purines in microplasmodia of Physarum polycephalum were investigated. The results revealed a unique pattern, namely that exogenous purine nucleosides are readily taken up and metabolised, while free purine bases are hardly taken up. The pathways of incorporation have been elucidated in studies with whole cells and with cell-free extracts. The ribonucleosides (adenosine, inosine and guanosine) can be converted into ribonucleotides in two ways; either directly catalysed by a kinase or by a phosphorolytic cleavage to the free base (adenine, hypoxanthine and guanine respectively) which can then be activated by a purine phosphoribosyltransferase. Apparently the purine phosphoribosyltransferases do not react with exogenous purine bases. The deoxyribonucleosides (deoxyadenosine, deoxyinosine and deoxyguanosine) are also phosphorolysed by purine nucleoside phosphorylase to adenine, hypoxanthine and guanine respectively. A portion of deoxyadenosine is directly phosphorylated to dAMP. It appears that only a minor part of the soluble nucleotide pool can be synthesised from exogenous supplied nucleosides and that none of the deoxyribonucleosides specifically label DNA. There is no catabolism of the purine moiety. In agreement with the above findings, we have found that analoguees of purine nucleosides are more toxic than their corresponding purine base analogues.     

Nucleoside Uptake by Slices of Mouse Brain

: The properties of the uptake of nucleosides and nucleotides by brain cells were examined in slices of mouse brain. Of the compounds tested, adenine and adenosine had the most rapid uptake and reached the highest levels. Uptake was mediated, as shown by saturability and strong inhibition, by low temperature, or by cyanide, and was only partially sodium- or calcium-dependent. The inhibition pattern by analogues indicated the presence of several uptake systems (possibly four), as shown by differences between adenine and guanine uptake, between adenine and adenosine uptake, and between adenosine and cytidine uptake. The properties of uptake systems for nucleotides and nucleosides were somewhat different from those for amino acids.     

Caffeine inhibition of aflatoxin production: mode of action.

Evaluation of caffeine and a number of related methylxanthines indicated that the ability of the compound to inhibit growth and aflatoxin production by Aspergillus parasiticus is highly specific and does not involve an inhibition of cyclic AMP phosphodiesterase. Supplementation of the culture medium with purine bases, nucleosides, and nucleotides suggested that the inhibition of fungal growth could be partially overcome by adenine or guanine but that the purines had little effect on the inhibition of aflatoxin production. Likewise, increasing the levels of trace minerals did not overcome the inhibition of toxin production. Electron microscopic evaluation of caffeine-treated and -untreated cultures indicated that the compound produced observable changes in the ultrastructure of the fungus.     

Incorporation of deoxyribonucleosides into DNA of coryneform bacteria and the relevance of deoxyribonucleoside kinases

In order to obtain basic knowledge of the salvage pathways for DNA synthesis, the ability of Brevibacterium ammoniagenes ATCC 6872 and Micrococcus luteus ATCC 15932 for incorporation of nucleobases and nucleosides was investigated. Only adenine and uracil are incorporated by B. ammoniagenes, whereas M. luteus additionally can utilize deoxyadenosine and, less efficiently, thymidine. In M. luteus, the demonstration of deoxyadenosine kinase and thymidine kinase explains the incorporation data. Uptake of thymidine is of short duration because of rapid breakdown of exogenously supplied thymidine to thymine. At a 540-fold excess pyrimidine deoxyribonucleosides inhibit 14C incorporation from thymidine nearly totally and purine deoxyribonucleosides cut by half the uptake rate, probably by interfering with transport of thymidine. However, as no cessation of thymidine incorporation occurs at these concentrations of purine deoxyribonucleosides, incorporation is finally enhanced. During the initial period of this reduced uptake considerable protection of thymidine from breakdown to thymine is provided by deoxyguanosine, but not by deoxyadenosine. At a 108-fold excess there is actually no inhibition of thymidine uptake by deoxyguanosine and only an insignificant impairment by deoxyadenosine resulting in an ultimate enhancement of 14C incorporation up to 20% of the exogenously supplied thymidine. As there is no salvage pathway for thymidine in B. ammoniagenes due to the absence of thymidine kinase, labelling with adenine and hydrolyzing of the 'contaminated' RNA fraction with 1 M KOH is recommended for measurements of overall DNA synthesis in this strain.     

Antibodies and autoantibodies of glycogen phosphorylase b: inactivation of pig and rabbit enzymes.

Pig skeletal muscle glycogen phosphorylase b was purified using ammonium sulfate fractionation, DEAE-Sephadex A-50 and Sephadex G-200 column chromatography. The purified enzyme was used to immunize rabbits in the presence or in the absence of complete Freund adjuvant. Antibodies against pig phosphorylase in pure form were isolated from rabbit antisera using insoluble immunoadsorbents of pig phosphorylase. Autoantibodies against the rabbit enzyme were obtained from the same antisera using insoluble immunoadsorbents of rabbit phosphorylase. Complete inactivation of pig phosphorylase was accomplished by an antibody/enzyme molar ratio equal to 4 and autoantibody/enzyme molar ratio equal to 130. Complete inactivation of rabbit phosphorylase was accomplished by an antibody/enzyme molar ratio equal to 250 and autoantibody/enzyme molar ratio equal to 160. Passive haemagglutination technique gave positive results with minimum amounts of 0.02 microng/ml and 0.8 microng/ml for pig and rabbit phosphorylase respectively. Kinetic experiments have shown that antibodies and autoantibodies act as noncompetitive inhibitors of both enzymes with respect to AMP and glucose 1-phosphate but exhibit a mixed type of inhibition with respect to glycogen. When glycogen hydrolysates were used as substrate in place of intact glycogen molecules a pronounced decrease in the inhibitory capacity of antienzyme on the enzyme was demonstrated.     

Relationship Between [8-14C]Adenosine Transport and Growth Inhibition in Neurospora crassa Strain ad-8

The kinetic parameters of [8-(14)C]adenosine transport by a general nucleoside uptake system were studied in germinated conidia of the ad 8 strain of Neurospora crassa. The apparent K(m) for adenosine uptake by this system was found to be 6.2 muM. The apparent K(i) values for other nucleosides competing with adenosine for uptake were measured by using Dixon plots. Nucleosides which were efficient competitive inhibitors of adenosine transport were found to inhibit severely the rate of growth of strain ad-8 on adenosine-supplemented medium. Xanthosine and thymidine did not inhibit [8-(14)C]adenosine uptake as severely as other nucleosides, nor did they cause significant inhibition of ad-8 growth rate on adenosine.     

Regulation of purine utilization in bacteria. VII. Involvement of membrane-associated nucleoside phosphorylase in the uptake and the base-mediated loss of the ribose moiety of nucleosides by Salmonella typhimurium membrane vesicles.

Although uridine and adenosine are converted by membrane-associated nucleoside phosphorylases to ribose-1-phosphate (ribose-1-P) and the corresponding bases (uracil and adenine), only ribose -1-P is accumulated within Salmonella typhimurium LT2 membrane vesicles. In accordance with these observations, no uptake is observed when the vesicles are incubated with the bases or nucleosides labeled in their base moieties. The vesicles lack a transport system for ribos-1-P, since excess ribose-1-P does not inhibit the uptake of the ribose moiety of uridine. In addition, there is no exchange with preaccumulatedribose-1-P. Thus, uridine, rather than ribose-1-P, must serve as the initially transported substrate. The uptake of the ribose portion of uridine is coupled to electron transport, and the levels to which ribose-1-P are accumulated may be reduced by adding various bases to the reaction mixtures. The bases appear to inhibit the uridine phosphorylase reaction and/or cause an efflux of ribose-1-P from the vesicles. This loss of ribose-1-P reflects the accumulation of nucleosides in the external medium after being synthesized within the membranes. Synthesis of the nucleosides from intravesicular ribose-1-P and exogenous base proceeds even though the bases are not accumulated by the vesicles. Furthermore, ribose-1-P cannot significantly inhibit uridine phosphorylase activity unless the membranes are disrupted. These observations indicate that the membrane-associated nucleoside phosphorylases may have a transmembranal orientation with their base and ribose-1-P binding sites on opposite sides of the membranes. Such an asymmetric arrangement of these enzymes may facilitate the uptake of the ribosyl moiety of nucleosides by a group translocation mechanism. Thus, nucleosides may be cleaved during the membrane transport process, with the resultant bases delivered to the external environment while ribose-1-P is shunted to the intravesicular space.     

Azione di Nucleosidi e Relative Basi su Alcuni Aspetti Fisiologici di Rhizobium Leguminosarum Frank

Abstract

On the action of some nucleosides and related bases on some physiologiacal aspects of RHIZOBIUM LEGUMINOSARUM Frank. — Research was carried out on the action of some nucleosides and related bases on some physiological aspects of Rhizobium leguminosarum Frank. The compounds tested were: adenine, adenosine, cytosine, guanine, guanosine, hypoxantine, inosine, thymine, thymidine, uracil, uridine.

Pyrimidinic nucleosides, apart from the disappearance of ribose, behave similarly to their bases.

The bases and nucleosides tested stimulate changes in the respiration of R. leguminosarum. Remarkably different values are observed in the endogenous respiration when compared to the control, and a remarkable delay in the beginning of respiration in the case of cells precultivated in presence of cytidine, uridine and thymidine. A number of these cells were later found to have an anaerobic metabolism, at least in the starting period. These findings may be explained admitting that the regulatory system of Rhizobium, with regard to oxygen uptake, are dependent on the presence of certain compunds, including some nucleosides, which the microorganism is in contact with within the nodule.

Apart from cytidine sulphate, there is a fundamental stimulatory effect on the growth of R. leguminosarum. After 24 hours incubation, purinic bases and their nucleosides are not detectable in cultural media. Thymine and cytosine, however, are still present in the medium, or have been transforned into uracil; the latter undergoes no transformation in the medium     

产胸苷磷酸化酶菌株初筛方法的建立及其应用

建立了一种快速简便地筛选高产胸苷磷酸化酶菌株的初筛方法及其在紫外诱变育种中的应用。结果显示,在液体培养基中,添加25mmol/L胸苷不会影响菌体生长且菌体中的胸苷磷酸化酶能催化胸苷生成胸腺嘧啶,在同等浓度下产物胸腺嘧啶OD_290nm远大于胸苷OD_290nm,从而导致发酵液OD_290nm显著升高,据此建立产胸苷磷酸化酶短乳杆菌的初筛方法。在此基础上结合紫外诱变条件的优化,确定初次紫外照射时间为20 s,停止5 min后再照射10 s、15 s、20 s,控制紫外致死率在80%左右,以此条件构建突变文库。通过初筛和复筛确定突变株EB₂₇、EA₄₂酶活分别达到1.025 U/mg湿菌体和0.916 U/mg湿菌体,较初始菌株提高了50%和35%,且具有良好的遗传稳定性。     

Transport of adenine, hypoxanthine and uracil into Escherichia coli.

Uptake of adenine, hypoxanthine and uracil by an uncA strain of Escherichia coli is inhibited by uncouplers or when phosphate in the medium is replaced by less than 1 mM-arsenate, indicating a need for both a protonmotive force and phosphorylated metabolites. The rate of uptake of adenine or hypoxanthine was not markedly affected by a genetic deficiency of purine nucleoside phosphorylase. In two mutants with undetected adenine phosphoribosyltransferase, the rate of adenine uptake was about 30% of that in their parent strain, and evidence was obtained to confirm that adenine had then been utilized via purine nucleoside phosphorylase. In a strain deficient in both enzymes adenine uptake was about 1% of that shown by wild-type strains. Uptake of hypoxanthine was similarly limited in a strain lacking purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase. Deficiency of uracil phosphoribosyltransferase severely limits uracil uptake, but the defect can be circumvented by addition of inosine, which presumably provides ribose 1-phosphate for reversal of uridine phosphorylase. The results indicate that there are porter systems for adenine, hypoxanthine and uracil dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases.     

Control of mammary gland fibroblast growth by insulin, growth hormone and prolactin

Fibroblasts isolated from guinea pig mammary glands were cultured in 96 well culture plates in the presence of various concentrations of insulin, growth hormone and prolactin. Insulin (30 micrograms/ml increased uptake of tritiated thymidine by 30%. Higher concentrations of insulin did not result in any further increase in thymidine uptake. Growth hormone alone did not alter thymidine uptake in concentrations of 0 to 250 ng/ml. 300 ng/ml gave thymidine uptake of 136% of controls. In the presence of 20 g/ml insulin, growth hormone (250 ng/ml) increased thymidine uptake to approximately double that of controls. Prolactin alone (300 ng/ml decreased thymidine uptake by 19%. Insulin increased thymidine uptake, but the negative effect of prolactin was still evident above 150 ng/ml.     

Biochemistry and metabolism of Giardia

Giardia lamblia, an aerotolerant anaerobe, respires in the presence of oxygen by a flavin, iron-sulfur protein-mediated electron transport system. Glucose appears to be the only sugar catabolized by the Embden-Meyerhof-Parnas and hexose monophosphate pathways, and energy is produced by substrate level phosphorylation. Substrates are incompletely oxidized to CO2, ethanol and acetate by nonsedimentable enzymes. The lack of incorporation of inosine, hypoxanthine, xanthine, formate or glycine into nucleotides indicates an absence of de novo purine synthesis. Only adenine, adenosine, guanine and guanosine are salvaged, and no interconversion of these purines was detected. Salvage of these purines and their nucleosides is accomplished by adenine phosphoribosyltransferase, adenosine hydrolase, guanosine phosphoribosyltransferase and guanine hydrolase. The absence of de novo pyrimidine synthesis was confirmed by the lack of incorporation of bicarbonate, orotate and aspartate into nucleotides, and by the lack of detectable levels of the enzymes of de novo pyrimidine synthesis. Salvage appears to be accomplished by the action of uracil phosphoribosyltransferase, uridine hydrolase, uridine phosphotransferase, cytidine deaminase, cytidine hydrolase, cytosine phosphoribosyltransferase and thymidine phosphotransferase. Nucleotides of uracil may be converted to nucleotides of cytosine by cytidine triphosphate synthetase, but thymidylate synthetase and dihydrofolate reductase activities were not detected. Uptake of pyrmidine nucleosides, and perhaps pyrimidines, appears to be accomplished by carrier-mediated transport, and the common site for uptake of uridine and cytidine is distinct from the site for thymidine. Thymine does not appear to be incorporated into nucleotide pools. Giardia trophozoites appear to rely on preformed lipids rather than synthesizing them de novo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purine metabolites suppress proliferation of human NK cells through a lineage-specific purine receptor.

NK cell proliferation is suppressed in some patients with cancer by unknown mechanisms. Because purine metabolites released into the extracellular space during cell lysis may affect cell function, we hypothesized that these metabolites could serve as feedback regulators of NK cell proliferation. Sorted NK (CD56+/CD3-) cells were incubated with IL-2 (1000 U/ml) in a 4-day thymidine uptake assay with or without 10-10,000 microM of nucleotides. Adenine nucleotides inhibited NK cell proliferation, with ATP = ADP > 5'-adenylylimidodiphosphate > AMP = adenosine; ADP-ribose and nicotinamide adenine dinucleotide, but not nicotinamide or UTP, caused a dose-dependent suppression of thymidine uptake. A total of 100 microM ATP, a concentration that induced a maximal (80%) inhibition of thymidine uptake, did not inhibit cytotoxic activity against K562 targets. Because NK cells retained the ability to lyse K562 targets 4 days after exposure to 500 microM ATP or 1000 microM adenosine, inhibition of thymidine uptake was not due to cell death. Incubation of NK cells with dibutyryl cAMP and forskolin also suppressed thymidine uptake. Cholera toxin and pertussis toxin suppressed NK cell proliferation. Pertussis toxin did not block the adenine nucleotide effects. Further, ATP, but not adenosine or other nucleotides, markedly increased intracellular cAMP in a dose-dependent manner. The ATP-induced increase in cAMP was specific to cytolytic cells, because CD19+ B cells and CD4+ T cells did not increase their intracellular cAMP. These studies demonstrate that NK proliferation is regulated through purine receptors by adenine nucleotides, which may play a role in decreased NK cell activity. The response to adenine nucleotides is lineage-specific.     

Enhanced thymidine uptake causes the lowered thymidine requirement of D. discoideum auxotroph HPS 401

Dictyostelium discoideum strain HPS 401 contains a spontaneous mutation that lowers the amount of thymidine required for cell growth relative to that of the auxotrophic parental strain HPS 400. Growth studies in defined medium show that as little as 8 micrograms thymidine/ml supports maximal growth of HPS 401, whereas 50 micrograms/ml is required by HPS 400. In contrast, both strains require over 40 micrograms thymidylate/ml to achieve maximal growth. HPS 401 exhibits thymidineless death when grown without thymidine; relative viability decreases to less than 0.01% after 190 h incubation. Assays for enzymes related to thymidine metabolism reveal that none of the strains tested (HPS 401, HPS 400, and prototrophic HPS 83 cells) contain detectable thymidine phosphorylase activity and that the specific activity of thymidine kinase is the same in these three strains. Thin-layer chromatography of extracts from cells grown on radiolabeled thymidine shows that there is no detectable conversion of thymidine to thymine in any of these strains. These analyses show that HPS 401 has rapid intracellular accumulation of thymidine, while only slight uptake is observed with HPS 400 or wild-type strains. HPS 401 also shows greater uptake of uridine in comparison to HPS 400 and wild-type cells. Thymidylate uptake was the same for all three strains. Thus, the mutation giving rise to the HPS 401 phenotype selectively increases the uptake of thymidine into the cell, where it can be efficiently utilized for DNA synthesis by the "salvage" pathways of nucleotide metabolism.     

Ribosyl and Deoxyribosyl Transfer by Bacterial Enzyme Systems

The enzymatic transfer of ribose and deoxyribose residues in pyrimidine nucleosides to purines was catalyzed by cell-free extracts of various bacteria. Almost all the strains belonging to Enterobacteriaceae were capable of catalyzing the transfer reactions. The transfer activities were also detected among some bacterial strains of other families: Pseudomonadaceae, Corynebacteriaceae, Micrococcaceae, Bacteriaceae, and Bacillaceae. The rates of the transfer reactions were greatly enhanced in the presence of phosphate ion, and the participation of nucleoside phosphorylases in the reactions was suggested. Uridine phosphorylase, thymidine phosphorylase, and purine nucleoside phosphorylase were purified from cell-free extract of Aerobacter aerogenes IFO 3321. The ribosyl transfer from uridine to hypoxanthine was found to be catalyzed by the coupled reactions of uridine and purine nucleoside phosphorylases and the deoxyribosyl transfer from thymidine to hypoxanthine by the coupled reactions of thymidine and purine nucleoside phosphorylases.