Purine Nucleoside Phosphorylase Activity Research Articles

Chemo-Enzymatic Generation of Highly Fluorescent Nucleoside Analogs Using Purine-Nucleoside Phosphorylase

Chemo-enzymatic syntheses of strongly fluorescent nucleoside analogs, potentially applicable in analytical biochemistry and cell biology are reviewed. The syntheses and properties of fluorescent ribofuranosides of several purine, 8-azapurine, and etheno-purine derivatives, obtained using various types of purine nucleoside phosphorylase (PNP) as catalysts, as well as α-ribose-1-phosphate (r1P) as a second substrate, are described. In several instances, the ribosylation sites are different to the canonical purine N9. Some of the obtained ribosides show fluorescence yields close to 100%. Possible applications of the new analogs include assays of PNP, nucleoside hydrolases, and other enzyme activities both in vitro and within living cells using fluorescence microscopy.

Engineering Substrate Promiscuity of Nucleoside Phosphorylase Via an Insertions-Deletions Strategy.

Nucleoside phosphorylases (NPs) are the key enzymes in the nucleoside metabolism pathway and are widely employed for the synthesis of nucleoside analogs, which are difficult to access via conventional synthetic methods. NPs are generally classified as purine nucleoside phosphorylase (PNP) and pyrimidine or uridine nucleoside phosphorylase (PyNP/UP), based on their substrate preference. Here, based on the evolutionary information on the NP-I family, we adopted an insertions-deletions (InDels) strategy to engineer the substrate promiscuity of nucleoside phosphorylase AmPNPΔS2V102K, which exhibits both PNP and UP activities from a trimeric PNP (AmPNP) of Aneurinibacillus migulanus. Furthermore, the AmPNPΔS2V102K exerted phosphorylation activities toward arabinose nucleoside, fluorosyl nucleoside, and dideoxyribose, thereby broadening the unnatural-ribose nucleoside substrate spectrum of AmPNP. Finally, six purine nucleoside analogues were successfully synthesized, using the engineered AmPNPΔS2V102K instead of the traditional "two-enzymes PNP/UP" approach. These results provide deep insights into the catalytic mechanisms of the PNP and demonstrate the benefits of using the InDels strategy to achieve substrate promiscuity in an enzyme, as well as broadening the substrate spectrum of the enzyme.

Study of changes in the activity levels of purine nucleoside phosphorylase and the role of nitric oxide in the development of pathology in alloxan-diabetic rats

The problem of treating diabetes mellitus has not been resolved to this day; it requires a detailed study of various links of pathogenesis at the molecular level. In diabetes, conditions arise for the formation of oxidative stress. Diabetes mellitus is associated with the early development of cardiovascular complications, the immune e system is destroyed. Endothelial cells produce nitric oxide (NO), a substance that is able to maintain a balance of vascular tone, coagulation and inflammation. Purine nucleoside phosphorylase (PNP) is one of the most important enzymes of purine metabolism, which characterizes the immune status of the organism. The aim of this study was to study the enzymatic activity of PNP, as well as to determine the levels of nitric oxide in the blood serum, liver and pancreas in albino rats with simulated alloxan diabetes. The results of our studies on the detection of changes in the activity of PNP in the pancreas on the model of alloxandiabetic rats showed that at the late stage, the activity of PNP in the pancreas is completely suppressed, which indicates significant changes in the immune status of the body, the content of nitric oxide increases. Understanding the complex metabolic disorders that interact with the NO system may provide us with additional therapeutic options to reduce cardiovascular morbidity and mortality in diabetes mellitus. Indicators of NO and PNP can be used as additional paraclinical indicators for early diagnosis and to identify and clarify the degree of activity of the pathological process, the nature of the course, the stage of the disease, which contributes to the appointment of individualized adequate therapy.

Lactiplantibacillus plantarum enables blood urate control in mice through degradation of nucleosides in gastrointestinal tract

BackgroundLactobacillus species in gut microbiota shows great promise in alleviation of metabolic diseases. However, little is known about the molecular mechanism of how Lactobacillus interacts with metabolites in circulation. Here, using high nucleoside intake to induce hyperuricemia in mice, we investigated the improvement in systemic urate metabolism by oral administration of L. plantarum via different host pathways.ResultsGene expression analysis demonstrated that L. plantarum inhibited the activity of xanthine oxidase and purine nucleoside phosphorylase in liver to suppress urate synthesis. The gut microbiota composition did not dramatically change by oral administration of L. plantarum over 14 days, indicated by no significant difference in α and β diversities. However, multi-omic network analysis revealed that increase of L. plantarum and decrease of L. johnsonii contributed to a decrease in serum urate levels. Besides, genomic analysis and recombinant protein expression showed that three ribonucleoside hydrolases, RihA–C, in L. plantarum rapidly and cooperatively catalyzed the hydrolysis of nucleosides into nucleobases. Furthermore, the absorption of nucleobase by intestinal epithelial cells was less than that of nucleoside, which resulted in a reduction of urate generation, evidenced by the phenomenon that mice fed with nucleobase diet generated less serum urate than those fed with nucleoside diet over a period of 9-day gavage.ConclusionCollectively, our work provides substantial evidence identifying the specific role of L. plantarum in improvement of urate circulation. We highlight the importance of the enzymes RihA–C existing in L. plantarum for the urate metabolism in hyperuricemia mice induced by a high-nucleoside diet. Although the direct connection between nucleobase transport and host urate levels has not been identified, the lack of nucleobase transporter in intestinal epithelial cells might be important to decrease its absorption and metabolization for urate production, leading to the decrease of serum urate in host. These findings provide important insights into urate metabolism regulation.BbgstkDZoHsJm_QsUh-oZNVideo

Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme.

Mammalian purine nucleoside phosphorylase (PNP) is biologically active as a homotrimer, in which each monomer catalyzes a reaction independently of the others. To answer the question of why the native PNP forms a trimeric structure, we constructed, in silico and in vitro, the monomeric form of the enzyme. Molecular dynamics simulations showed different geometries of the active site in the non-mutated trimeric and monomeric PNP forms, which suggested that the active site in the isolated monomer could be non-functional. To confirm this hypothesis, six amino acids located at the interface of the subunits were selected and mutated to alanines to disrupt the trimer and obtain a monomer (6Ala PNP). The effects of these mutations on the enzyme structure, stability, conformational dynamics, and activity were examined. The solution experiments confirmed that the 6Ala PNP mutant occurs mainly as a monomer, with a secondary structure almost identical to the wild type, WT PNP, and importantly, it shows no enzymatic activity. Simulations confirmed that, although the secondary structure of the 6Ala monomer is similar to the WT PNP, the positions of the amino acids building the 6Ala PNP active site significantly differ. These data suggest that a trimeric structure is necessary to stabilize the geometry of the active site of this enzyme.

Purine Nucleoside Phosphorylase Inhibition Attenuates the Progression of Anemia and Organ Damage in Sickle Cell Mice

Purine nucleoside phosphorylase (PNP) converts guanosine to guanine, inosine to hypoxanthine, and 2'-deoxyguanosine to guanine, and leads to the downstream formation of the pro-oxidant and vasculotoxic hypoxanthine and xanthine. Red blood cells are rich in PNP, yet PNP's role in the pathogenesis of sickle cell disease (SCD) is not known. Our group showed that patients with SCD have increased PNP release and activity, leading to accelerated guanosine/inosine metabolism and increased production of vasculotoxic hypoxanthine/xanthine (Bilan VP et al. 2018). Thus, we proposed that PNP may have a major pathogenic role in red blood cell sickling, hemolytic angioproliferation, cell aggregation and end organ damage, and that PNP inhibition may be beneficial in the treatment of SCD. The highly potent PNP inhibitor 8-aminoguanine is the product of the conversion of the endogenous guanosine metabolite and pro-drug 8-aminoguanosine (8-AG). We investigated the safety and efficacy of short-term (8 weeks) and chronic (20 weeks) treatment of Townes sickle mice (SS) and their non-sickling controls (AA) with 8-AG (60mg/kg/day administered ad libitum in the drinking water). We collected blood and urine samples at multiple intervals using metabolic cages. To determine 8-AG body disposition and PNP inhibition, urine samples were analyzed for purines by liquid chromatography - tandem mass spectrometry (LC-MS/MS). Both AA and SS mice tolerated 8-AG well with no evidence of toxicity. In both AA (n= 15) and SS (n=40) mice there was very efficient conversion of 8-AG that resulted in a 40-50-fold increase in urinary 8-aminoguanine concentrations (p<0.001). In 8-AG treated SS mice we observed reduced hypoxanthine levels, with an increased inosine/hypoxanthine ratio. Additionally, guanosine was increased, guanine was reduced, and an increased guanosine/guanine ratio was observed (p<0.05), suggesting potent PNP inhibition. In SS mice, the 8-week treatment with 8-AG significantly reduced hemoglobinuria (p<0.05) and albuminuria (p<0.05), attenuated left ventricular (LV, p<0.01) and right ventricular (RV, p<0.05) hypertrophy, limited hepatomegaly (p<0.001), and improved RV contractility (p<0.05), with a trend towards reduced lung weights and splenomegaly. In SS mice, 20-week treatment with 8-AG significantly slowed the progression of anemia as evidenced by attenuated age-related drops in hemoglobin and hematocrit levels, and it attenuated hemoglobinuria and albuminuria while significantly reducing splenomegaly (p=<0.05) and hepatomegaly (p<0.05, Figure). Semi-quantitative histopathological analysis suggested reduced iron deposition (Prussian blue stain, Figure), and reduced ischemic (H&E stain) and fibrotic (trichrome stain, Figure) injury in the spleen and liver from SS mice treated with 8-AG. Our data suggest that the PNP inhibitor 8-AG may have a pharmacological benefit in SCD and may warrant further investigation as a multi-pronged treatment approach for SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Purine nucleoside phosphorylase controls nicotinamide riboside metabolism in mammalian cells

Nicotinamide riboside (NR) is an effective precursor of nicotinamide adenine dinucleotide (NAD) in human and animal cells. NR supplementation can increase the level of NAD in various tissues and thereby improve physiological functions that are weakened or lost in experimental models of aging or various human pathologies. However, there are also reports questioning the efficacy of NR supplementation. Indeed, the mechanisms of its utilization by cells are not fully understood. Herein, we investigated the role of purine nucleoside phosphorylase (PNP) in NR metabolism in mammalian cells. Using both PNP overexpression and genetic knockout, we show that after being imported into cells by members of the equilibrative nucleoside transporter family, NR is predominantly metabolized by PNP, resulting in nicotinamide (Nam) accumulation. Intracellular cleavage of NR to Nam is prevented by the potent PNP inhibitor Immucillin H in various types of mammalian cells. In turn, suppression of PNP activity potentiates NAD synthesis from NR. Combining pharmacological inhibition of PNP with NR supplementation in mice, we demonstrate that the cleavage of the riboside to Nam is strongly diminished, maintaining high levels of NR in blood, kidney, and liver. Moreover, we show that PNP inhibition stimulates Nam mononucleotide and NAD+ synthesis from NR invivo, in particular, in the kidney. Thus, we establish PNP as a major regulator of NR metabolism in mammals and provide evidence that the health benefits of NR supplementation could be greatly enhanced by concomitant downregulation of PNP activity.

P1312: FORODESINE AMPLIFIES HOST INNATE IMMUNE RESPONSE THROUGH TOLL-LIKE RECEPTOR 7 ACTIVATION WHILE PREVENTING EXPERIMENTAL GRAFT-VERSUS-HOST DISEASE

Background: Forodesine, a novel inhibitor of purine nucleoside phosphorylase (PNP), causes nucleotide pool imbalances by accumulating intracellular deoxyguanosine, thereby resulting in selective apoptosis of T-cells. Recent evidence indicates that specific types of nuclear acids including guanosine and its derivatives act as natural ligands for toll-like receptor 7 (TLR7). So far, it is unclear whether and how forodesine can modulate host innate immunity, including antigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs), which express TLR7 abundantly. Aims: The aim of this study is to investigate the role of forodesine in host APC function as well as donor T-cell activation both in vivo graft-versus-host disease (GVHD) models and in vitro cell assays. Methods: NOG mice were subjected to 250 cGy total body irradiation followed by intravenous injection of human isolated T-cells. Mice were treated with forodesine (20 mg/kg) or vehicle daily after transplantation. Results: Despite the mice without treatment developing severe GVHD after transplantation, guanosine and deoxyguanosine were hardly detected in the plasma, suggesting that purine nucleosides released from damaged tissues were rapidly degraded by PNP. In fact, inhibition of PNP activity by forodesine markedly increased plasma guanosine levels, whereas deoxyguanosine was still undetectable. This was also the case in GVHD target organs, including liver and lungs. At the same level of guanosine in the liver, forodesine promoted TNF-a production from in vitro alveolar macrophage cultures. Preincubation with a TLR7 antagonist, ODN20958, attenuated the TNF-a production from macrophages stimulated by guanosine and forodesine. Nuclease treatment did not reduce macrophage TNF-a production, suggesting that single-strand RNA, a well-known TLR7 ligand, is not involved in this response. Similar results were observed in Flt3-ligand-induced plasmacytoid DCs. Not only TNF-a but also IL-12 production was induced by the combination of guanosine and forodesine, and was comparable to that in pDCs pulsed with R848, a potent agonist of TLR7/8. These observations suggest that a sustained high level of guanosine caused by forodesine treatment lead to continuous activation of TLR7 signaling, resulting in cytokine production from APCs. Meanwhile, as reported previously, forodesine inhibited the proliferation of human T-cells activated with CD3 and CD28 antibodies in the presence of deoxyguanosine. Of note, however, this combination did not impair but rather stimulated IFN-g production from activated T-cells cocultured with pDCs. This suggests that T-cell priming capacity mediated by the interaction with pDCs is conserved through TLR7 activation. Finally, we investigated the in vivo effects of forodesine on survival, body weight change, and disease severity of GVHD mice. Forodesine treatment significantly prolonged survival of the mice compared with control mice, with a median survival of 17.5 days vs 14 days, respectively (p = 0.029). The mean body weight loss percentage at day 9 was 11.5% vs 19.0% in the forodesine group and control, respectively (p = 0.003). Furthermore, the clinical GVHD scores were also lower in the forodesine group vs control (4.9 vs 0.6 points, p = 0.001). Image:Summary/Conclusion: This study demonstrates that forodesine amplifies host innate immune response through TLR7 signaling, while ameliorating T-cell GVH reaction. Further studies are needed to investigate whether forodesine can provide host protection against infections in a transplant setting.

A Direct OnFlow Assay to Monitor the Activity of Purine Nucleoside Phosphorylase from Mycobacterium tuberculosis

A Direct OnFlow Assay to Monitor the Activity of Purine Nucleoside Phosphorylase from Mycobacterium tuberculosis

Gender difference in arsenic biotransformation is an important metabolic basis for arsenic toxicity

BackgroundArsenic metabolism enzymes can affect the toxic effects of arsenic. However, the effects of different genders on the metabolites and metabolic enzymes in liver arsenic metabolism is still unclear. This study analyzed the gender differences of various arsenic metabolites and metabolic enzymes and further explored the effects of gender differences on arsenic metabolism in liver tissues of rats.MethodsRats were treated with high/medium/low doses of iAs3+ or iAs5+. Liver pathological changes were observed with electron microscopy. The monomethyl aracid (MMA) and dimethyl aracid (DMA) was determined by high performance liquid chromatography-hydride generation atomic fluorescence spectroscopy. S-adenosylmethionine (SAM), arsenate respiratory reductase (ARR), nicotinamide adenine dinucleotide (NAD), purine nucleoside phosphorylase (PNP), pyruvate kinase (PK), and myeloperoxidase (MPO) SAM, ARR, NAD, PNP, PK, and MPO were determined by enzyme-linked immunoassay. RT-qPCR was used to determine Arsenic (+ 3 oxidation state) methyltransferase (AS3MT).ResultsThe iAs3+ and iAs5+ at high doses induced pathological changes in the liver, such as increased heterochromatin and lipid droplets. Compared within the same group, MMA and DMA were statistically significant in iAs3 + high, iAs3 + medium and iAs5+ low dose groups (P < 0.05). MMA of male rats in iAs3+ high and medium groups was higher than that of female rats, and the DMA of male rats was lower than that of female rats. As3MT mRNA in the male iAs3+ high group was higher than that of females. Besides, compared between male and female, only in iAS3+ low dose, iAS3+ medium dose, iAS5+ low dose, and iAS5+ medium dose groups, there was significant difference in SAM level (P < 0.05). Compared within the same group, male rats had significantly higher PNP and ARR activities while lower PK activity than female rats (P < 0.05). Between the male and female groups, only the iAS3+ high dose and medium dose group had a statistically significant difference (P < 0.05). The NAD activity of females in iAS3+ high dose group was higher than that of males.ConclusionThe gender differences in the arsenic metabolism enzymes may affect the biotransformation of arsenic, which may be one of the important mechanisms of arsenic toxicity of different sexes and different target organs.

Site-directed mutation of purine nucleoside phosphorylase for synthesis of 2'-deoxy-2'-fluoroadenosine

Nucleoside analogs are commonly used drugs for the treatment of cancer and viral infections. Purine nucleoside phosphorylase (PNP) is one of key enzymes required for the biosynthesis of 2’-deoxy-2’-fluoroadenosine. To improve PNP activity, two methods for selecting mutation sites were used based on molecular docking and dynamic simulations of PNP and various substrates. An efficient PNP mutant screening method was established. The variant E166 F/M167D 2 M had the highest activity and was selected for further characterization. The enzymatic activity and reaction rate of transglycosylation catalyzed by this variant were increased by 47.6 % and 38.8 % compared with the wild-type PNP, respectively. Two representative variants were used for analysis of conformational differences. We discovered that the flexibility of the random coil where Phe159 was located had a significant impact on the active center. In addition, the synthesis of 2’-deoxy-2’-fluoroadenosine was scaled up using 500 mL of phosphate buffer (100 mM, pH 7.0) containing 62.5 mM adenine, 25 mM 2’-deoxy-2’-fluorouridine, 3 mg thymidine phosphorylase (TP), and 3 mg E166 F/M167D 2 M. The concentration of the product reached 14.35 mM and the conversion rate reached 57.4 %. Thus, this process represents a promising approach for industrial production of 2’-deoxy-2’-fluoroadenosine.

Effects of quercetin and metabolites on uric acid biosynthesis and consequences for gene expression in the endothelium

BackgroundUric acid, a metabolic product of purine degradation in humans, is a risk factor for developing gout and type 2 diabetes, and supplementation with quercetin lowers plasma uric acid in mildly hyperuricemic men. Here we examined the mechanism of inhibition of enzymes involved in uric acid metabolism by quercetin, conjugates and microbial catabolites, and measured the effect of lowered circulating uric acid on endothelial cell gene expression. MethodsInhibition of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and xanthine oxidoreductase (XOR) activity by quercetin and metabolites was determined by HPLC. Human umbilical vein endothelial cells (HUVECs) were cultured under conditions mimicking blood flow, treated with uric acid (0, 300 or 500 μmol/L), and changes in gene expression measured using transcriptomics and quantitative droplet digital PCR. ResultsIn human plasma, no inhibition of PNP activity was observed, and only quercetin weakly inhibited ADA. XOR was not present at sufficient amount in human plasma to use for testing, but quercetin, quercetin-3′-sulfate and the gut microbial metabolite 3′,4′-dihydroxyphenylacetic acid inhibited bovine milk XOR. Several changes were observed in gene expression in HUVECs under flow compared to static conditions, but after uric acid treatment, only very few changes were detected. ConclusionsWe propose that the main mechanism by which quercetin, as quercetin-3′-sulfate, lowers uric acid in vivo is through inhibition of XOR, and not ADA nor PNP. The pertinent shift in uric acid concentration was not sufficient to produce significant changes in endothelial gene expression in a cell model.

Purine Nucleoside Phosphorylase (PNP) Deficiency: A Case Report

Background: Purine nucleoside phosphorylase (PNP) deficiency is a rare genetic disease that results in combined immunodeficiency. Its inheritance is autosomal recessive and affects the purine metabolic pathway. There is a profound effect on T-cells and variable B-cell dysfunction. There is a strong association with neurological dysfunction in as many as two thirds of cases and autoimmunity in one third. When PNP activity is absent or greatly diminished, deoxyguanosine triphosphate (dGTP) is believed to accumulate in the mitochondria, which inhibits ribonucleotide reductase and mitochondrial DNA repair. This is very harmful to T lymphocytes and leads to DNA damage and apoptosis during thymus selection. Case: A toddler girl diagnosed with PNP) deficiency that is currently being managed using different type of modalities with good response. Results: The patient was admitted in an outside hospital when she developed fever for 4 days along with a perianal abscess. Septic shock was suspected, and she was initially started on gentamycin and tazocin then switched to vancomycin, mereponem and metrodinazole. After resolution of fever, the patient was transferred to our tertiary care center to undergo immunology work up. Upon finally receiving the patient she was stable and afebrile, however there was severe neutropenia and leukopenia. A multidisciplinary team, which included general pediatrics, hematology, Infectious diseases and immunology, handled her case. Furthermore, the patient was switched back to tazocin and gentamycin and completed 14 days with good response and full resolution of fever. Following the multidisciplinary team plan, an immunology/hematology work up was initiated. Conclusion: After 14 days of treatment, there was a good response and resolution of fever. Then, a multidisciplinary team plan, an immunology/hematology work up was initiated.

Non-typical nucleoside analogs as fluorescent and fluorogenic indicators of purine-nucleoside phosphorylase activity in biological samples

Several novel non-typical nucleoside analogs were examined as potential fluorescent indicators of purine-nucleoside phosphorylase (PNP) activity in human blood. The substrates included N7-riboside of 8-aza-2,6-diaminopurine, N6-riboside of 1,N6-etheno-adenine and N2-riboside of N2,3-etheno-2-aminopurine. Reaction rates and apparent Michaelis’ constants were determined in 1000-fold blood lysates and compared with those for reference compounds, guanosine and 7-methylguanosine. It was concluded that the most promising for assaying human PNP in biological material was N6-riboside of 1,N6-etheno-adenine and N2-riboside of N2,3-etheno-2-aminopurine was optimal for the E. coli PNP, both offering at least 10-fold improvement in sensitivity relative to conventional assays. Other potential applications of this approach are discussed.

Энзимное профилирование плазмы крови и лимфоцитов при системной красной волчанке: фокус на ферменты пуринового и пиримидинового метаболизма

Изучение метаболизма пуриновых и пиримидиновых оснований при системной красной волчанке в настоящее время является актуальным направлением, как с позиций фундаментальной науки, так и в аспекте разработки инновационных средств лечения. Не менее важным является и энзимное профилирование, поскольку именно ферменты - перспективные предикторы ответа на терапию и удобные «мишени» для терапевтических воздействий. Цель исследования - описание профиля активности ключевых ферментов пуринового и пиримидинового метаболизма в плазме крови и в лизатах циркулирующих лимфоцитов у больных системной красной волчанкой. Методика. В плазме крови и лизатах лимфоцитов 50 больных системной красной волчанкой определяли активность комплекса из 10 ферментов пуринового и пиримидинового метаболизма. В качестве контроля использовали образцы 30 здоровых лиц. Оценка степени активности проводилась с использованием шкалы индексов ECLAM. Выделение лимфоцитов из венозной периферической крови проводили c помощью метода седиментации в градиенте плотности. Результаты. У больных системной красной волчанкой была установлена зависимость активности ферментов пуринового пиримидинового метаболизма от тяжести заболевания. Прямые корреляционные связи с индексом ECLAM выявлены в плазменной активности пуриннуклеозидфосфорилазы, аденозинкиназы, урацил/тимидиндегидрогеназы, ИМФ-дегидрогеназы, цитидиндезаминазы, тимидинкиназы, дигидрооротатдегидрогеназы, а также у активности аденозинкиназы, ИМФ-дегидрогеназы и тимидинкиназы в лизатах лимфоцитов. Обратные корреляционные связи с индексом ECLAM выявлены для аденозиндезаминазы, тимидинфосфорилазы в плазме и активности пуриннуклеозидфосфорилазы, аденозиндезаминазы, гуанилаткиназы, урацил/тимидиндегидрогеназы, цитидиндезаминазы, тимидинфосфорилазы, дигидрооротатдегидрогеназы в лизатах лимфоцитов. При системной красной волчанке в плазме наиболее информативными оказались показатели минимальной активности пуриннуклеозидфосфорилазы, аденозинкиназы и ИМФ-дегидрогеназы, в лимфоцитах - пуриннуклеозидфосфорилазы, аденозиндезаминазы и аденозинкиназы. Заключение. Изученные энзимные показатели можно использовать в качестве дополнительных маркеров активности системной красной волчанке. Purine and pyrimidine metabolic pathways are emerging topical areas of research from the perspective of both basic science and development of innovative therapies for systemic lupus erythematosus (SLE). It is particularly important, therefore, to disclose characteristic enzymatic patterns for further prediction of the response to treatment. Objective: to characterize activity patterns of the major enzymes of purine and pyrimidine metabolic pathways in SLE. Methods. Samples were obtained from 50 patients with verified SLE and 30 healthy controls. Disease activity was assessed using the ECLAM scale. Blood lymphocytes were isolated by a standard density gradient centrifugation procedure. Activities of 10 major purine and pyrimidine enzymes were measured in blood plasma and lysed lymphocytes. Results. For different enzyme groups, enzyme activities directly or inversely correlated with SLE severity. Plasma purine nucleoside phosphorylase, adenosine kinase, uracil/thymidine dehydrogenase, IMP dehydrogenase, cytidine deaminase, thymidine kinase, dihydroorotate dehydrogenase, and lymphocyte adenosine kinase, IMP dehydrogenase, and thymidine kinase activities positively correlated with the ECLAM score. Negative correlations with ECLAM score were found for plasma adenosine deaminase and thymidine phosphorylase, and for lymphocyte purine nucleoside phosphorylase, adenosine deaminase, guanylate kinase, uracil/thymidine dehydrogenase, cytidine deaminase, thymidine phosphorylase, and dihydroorotate dehydrogenase. Activities of plasma purine nucleoside phosphorylase, adenosine kinase, IMP dehydrogenase, and lymphocyte purine nucleoside phosphorylase, adenosine deaminase, and adenosine kinase activities had the highest correlations with minimal SLE activity and represented candidate markers for the disease severity. Conclusion. The studied enzymatic patterns can be used as auxiliary markers of SLE activity, with special emphasis on minimal disease activity.

Experimental intravascular hemolysis induces hemodynamic and pathological pulmonary hypertension: association with accelerated purine metabolism.

Pulmonary hypertension (PH) is emerging as a serious complication associated with hemolytic disorders, and plexiform lesions (PXL) have been reported in patients with sickle cell disease (SCD). We hypothesized that repetitive hemolysis per se induces PH and angioproliferative vasculopathy and evaluated a new mechanism for hemolysis-associated PH (HA-PH) that involves the release of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) from erythrocytes. In healthy rats, repetitive administration of hemolyzed autologous blood (HAB) for 10 days produced reversible pulmonary parenchymal injury and vascular remodeling and PH. Moreover, the combination of a single dose of Sugen-5416 (SU, 200 mg/kg) and 10-day HAB treatment resulted in severe and progressive obliterative PH and formation of PXL (Day 26, right ventricular peak systolic pressure (mmHg): 26.1 ± 1.1, 41.5 ± 0.5 and 85.1 ± 5.9 in untreated, HAB treated and SU+HAB treated rats, respectively). In rats, repetitive administration of HAB increased plasma ADA activity and reduced urinary adenosine levels. Similarly, SCD patients had higher plasma ADA and PNP activity and accelerated adenosine, inosine, and guanosine metabolism than healthy controls. Our study provides evidence that hemolysis per se leads to the development of angioproliferative PH. We also report the development of a rat model of HA-PH that closely mimics pulmonary vasculopathy seen in patients with HA-PH. Finally, this study suggests that in hemolytic diseases released ADA and PNP may increase the risk of PH, likely by abolishing the vasoprotective effects of adenosine, inosine and guanosine. Further characterization of this new rat model of hemolysis-induced angioproliferative PH and additional studies of the role of purines metabolism in HA-PH are warranted.

Purine-related metabolites and their converting enzymes are altered in frontal, parietal and temporal cortex at early stages of Alzheimer's disease pathology.

Adenosine, hypoxanthine, xanthine, guanosine and inosine levels were assessed by HPLC, and the activity of related enzymes 5'-nucleotidase (5'-NT), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) measured in frontal (FC), parietal (PC) and temporal (TC) cortices at different stages of disease progression in Alzheimer's disease (AD) and in age-matched controls. Significantly decreased levels of adenosine, guanosine, hypoxanthine and xanthine, and apparently less inosine, are found in FC from the early stages of AD; PC and TC show an opposing pattern, as adenosine, guanosine and inosine are significantly increased at least at determinate stages of AD whereas hypoxanthine and xanthine levels remain unaltered. 5'-NT is reduced in membranes and cytosol in FC mainly at early stages but not in PC, and only at advanced stages in cytosol in TC. ADA activity is decreased in AD when considered as a whole but increased at early stages in TC. Finally, PNP activity is increased only in TC at early stages. Purine metabolism alterations occur at early stages of AD independently of neurofibrillary tangles and β-amyloid plaques. Alterations are stage dependent and region dependent, the latter showing opposite patterns in FC compared with PC and TC. Adenosine is the most affected of the assessed purines.

Intracellular rebinding of transition-state analogues provides extended in vivo inhibition lifetimes on human purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) is part of the human purine salvage pathway. Its deficiency triggers apoptosis of activated T-cells, making it a target for T-cell proliferative disorders. Transition-state analogues of PNP bind with picomolar (pm) dissociation constants. Tight-binding PNP inhibitors show exceptionally long lifetimes on the target enzyme. We solve the mechanism of the target residence time by comparing functional off-rates in vitro and in vivo We report in vitro PNP-inhibitor dissociation rates (t½) from 3 to 31 min for seven Immucillins with dissociation constants of 115 to 6 pm Treatment of human erythrocytes with DADMe-Immucillin-H (DADMe-ImmH, 22 pm) causes complete inhibition of PNP. Loss of [14C]DADMe-ImmH from erythrocytes during multiple washes is slow and biphasic, resulting from inhibitor release and rebinding to PNP catalytic sites. The slow phase gave a t½ of 84 h. Loss of [14C]DADMe-ImmH from erythrocytes in the presence of excess unlabeled DADMe-ImmH increased to a t½ of 1.6 h by preventing rebinding. Thus, in human erythrocytes, rebinding of DADMe-ImmH is 50-fold more likely than diffusional loss of the inhibitor from the erythrocyte. Humans treated with a single oral dose of DADMe-ImmH in phase 1 clinical trials exhibit regain of PNP activity with a t½ of 59 days, corresponding to the erythropoiesis rate in humans. Thus, the PNP catalytic site recapture of DADMe-ImmH is highly favored in vivo We conclude that transition-state analogues with picomolar dissociation constants exhibit long lifetimes on their targets in vivo because the probability of the target enzyme recapturing inhibitor molecules is greater than diffusional loss to the extracellular space.

Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening

A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237–0.833 U/ml and 9.013–10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity.

Development of a new HPLC method using fluorescence detection without derivatization for determining purine nucleoside phosphorylase activity in human plasma.

Purine nucleoside phosphorylase (PNP) activity is involved in cell survival and function, since PNP is a key enzyme in the purine metabolic pathway where it catalyzes the phosphorolysis of the nucleosides to the corresponding nucleobases. Its dysfunction has been found in relevant pathological conditions (such as inflammation and cancer), so the detection of PNP activity in plasma could represent an attractive marker for early diagnosis or assessment of disease progression. Thus the aim of this study was to develop a simple, fast and sensitive HPLC method for the determination of PNP activity in plasma. The separation was achieved on a Phenomenex Kinetex PFP column using 0.1% formic acid in water and methanol as mobile phases in gradient elution mode at a flow rate of 1ml/min and purine compounds were detected using UV absorption and fluorescence. The analysis was fast since the run was achieved within 13min. This method improved the separation of the different purines, allowing the UV-based quantification of the natural PNP substrates (inosine and guanosine) or products (hypoxanthine and guanine) and its subsequent metabolic products (xanthine and uric acid) with a good precision and accuracy. The most interesting innovation is the simultaneous use of a fluorescence detector (excitation/emission wavelength of 260/375nm) that allowed the quantification of guanosine and guanine without derivatization. Compared with UV, the fluorescence detection improved the sensitivity for guanine detection by about 10-fold and abolished almost completely the baseline noise due to the presence of plasma in the enzymatic reaction mixture. Thus, the validated method allowed an excellent evaluation of PNP activity in plasma which could be useful as an indicator of several pathological conditions.