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James R. Hammond - One of the best experts on this subject based on the ideXlab platform.
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Pharmacological analysis and molecular cloning of the canine equiliBrative nucleoside transporter 1
European journal of pharmacology, 2004Co-Authors: James R. Hammond, Meaghan Stolk, Richard G. E. Archer, Kristy McconnellAbstract:ABstract We studied the Binding of [ 3 H]nitroBenzylthioinosine (NBMPR) and the uptake of [ 3 H]<B>FormycinB> B By the es (equiliBrative inhiBitor-sensitive) nucleoside transporter of Madin DarBy Canine Kidney (MDCK) cells. NBMPR inhiBited [ 3 H]<B>FormycinB> B uptake with a K i of 2.7±0.6 nM, and [ 3 H]NBMPR had a K D of 1.3±0.3 nM for Binding to these cells; these values are significantly higher than those oBtained in human and mouse cell models. In contrast, other recognized es inhiBitors, such as dipyridamole, were significantly more effective as inhiBitors of [ 3 H]NBMPR Binding and [ 3 H]<B>FormycinB> B uptake By MDCK cells relative to that seen for human cells. We isolated a cDNA encoding the canine es nucleoside transporter (designated cENT1), and assessed its function By staBle expression in nucleoside transport deficient PK15NTD cells. The PK15-cENT1 cells displayed inhiBitor sensitivities that were comparaBle to those oBtained for the endogenous es nucleoside transporter in MDCK cells. These data indicate that the dog es /ENT1 transporter has distinctive inhiBitor Binding characteristics, and that these characteristics are a function of the protein structure as opposed to the environment in which it is expressed.
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[3H]gemcitaBine uptake By nucleoside transporters in a human head and neck squamous carcinoma cell line.
The Journal of pharmacology and experimental therapeutics, 1999Co-Authors: James R. Hammond, Stephanie Lee, Peter J. FergusonAbstract:Cellular uptake of many chemotherapeutic nucleoside analogs is dependent on the activity of a family of nucleoside transport proteins located in the cell plasma memBrane. In the present study, we examined the role of these transporters in the accumulation of gemcitaBine By a human head and neck squamous carcinoma cell line. The uptake of [3H]gemcitiBine was compared with that of [3H]uridine and [3H]<B>FormycinB> B in the parent cell line (HN-5a) and in a gemcitaBine-resistant variant (GEM-8e). The HN-5a and GEM-8e cells were similar in their transport characteristics and expressed predominantly the es (equiliBrative, inhiBitor-sensitive) transporter suBtype; less than 10% of the influx of [3H]<B>FormycinB> B or [3H]uridine was mediated By the ei (equiliBrative inhiBitor-resistant) system, and there was no evidence for Na+-dependent nucleoside transporters. [3H]GemcitaBine (10 μM) entered these cells via Both the es and ei transporters with an initial rate of uptake similar to that seen with the use of [3H]<B>FormycinB> B or [3H]uridine. In addition, ATP-replete cells accumulated significantly less [3H]gemcitaBine than did ATP-depleted cells, which is indicative of an active efflux mechanism for gemcitaBine. These results show that gemcitaBine is a suBstrate for Both the es and ei nucleoside transporters of HN-5a and GEM-8e cells and that gemcitaBine resistance of the GEM-8e cells cannot Be attriButed to changes in transporter activity. Further studies to define the characteristics of the putative efflux mechanism are clearly warranted Because this system has the potential to significantly affect the clinical efficacy of gemcitaBine.
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interaction of 2 2 difluorodeoxycytidine gemcitaBine and <B>FormycinB> B with the na dependent and independent nucleoside transporters of ehrlich ascites tumor cells
Journal of Pharmacology and Experimental Therapeutics, 1998Co-Authors: Trisha Burke, Stephanie Lee, Peter J. Ferguson, James R. HammondAbstract:The uptake of [3H]<B>FormycinB> B By Ehrlich ascites tumor cells was examined in Both normal Na+ Buffer (physiological) and nominally Na+-free Buffer (iso-osmotic replacement with Li+). These studies were conducted to further characterize the equiliBrative nucleoside transporter suBtypes of Ehrlich cells and to assess the contriBution of Na+-dependent concentrative transport mechanisms to the cellular accumulation of nucleoside analogues By these cells. <B>FormycinB> B is poorly metaBolized By mammalian cells and, hence, can Be used as a suBstrate to measure transport kinetics in energetically competent cells. Initial studies estaBlished that <B>FormycinB> B inhiBited [3H]uridine uptake By the ei (equiliBrative inhiBitor-insensitive) and es (equiliBrative inhiBitor-sensitive) transporters of Ehrlich cells with Ki values of 48 +/- 28 and 277 +/- 25 microM, respectively. Similarly, [3H]<B>FormycinB> B had Km values of 111 +/- 52 and 635 +/- 147 microM for uptake By the ei and es transporters, respectively. When assays were conducted in the presence of Na+, plus 100 nM nitroBenzylthioinosine to prevent efflux via the es transporters, the intracellular concentration of [3H]<B>FormycinB> B exceeded the initial medium concentration By more than 3-fold, indicating the activity of a Na+-dependent transporter. Interestingly, the initial rate of uptake of [3H]<B>FormycinB> B was significantly higher in the Li+ Buffer (es-mediated Vmax = 65 +/- 10 pmol/microliter . sec) than in the Na+ Buffer (Vmax = 8.4 +/- 0.9 pmol/microliter . sec); this may reflect trans-acceleration of [3H]<B>FormycinB> B uptake By elevated intracellular adenosine levels resulting from the low Na+ environment. This model was then used to assess the interaction of gemcitaBine (2',2'-difluorodeoxycytidine) with the equiliBrative and concentrative nucleoside transporters. GemcitaBine, which has shown consideraBle potential for the treatment of solid tumors, was a relatively poor inhiBitor of [3H]<B>FormycinB> B uptake via the equiliBrative transporters (IC50 approximately 400 microM). In contrast, gemcitaBine was a potent inhiBitor of the Na+-dependent nucleoside transporter of Ehrlich cells (IC50 = 17 +/- 5 nM). These results suggest that the cellular expression/activity of Na+-dependent nucleoside transporters may Be an important determinant in gemcitaBine cytotoxicity and clinical efficacy.
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Interaction of 2′,2′-Difluorodeoxycytidine (GemcitaBine) and <B>FormycinB> B with the Na + -Dependent and -Independent Nucleoside Transporters of Ehrlich Ascites Tumor Cells
The Journal of pharmacology and experimental therapeutics, 1998Co-Authors: Trisha Burke, Stephanie Lee, Peter J. Ferguson, James R. HammondAbstract:The uptake of [3H]<B>FormycinB> B By Ehrlich ascites tumor cells was examined in Both normal Na+ Buffer (physiological) and nominally Na+-free Buffer (iso-osmotic replacement with Li+). These studies were conducted to further characterize the equiliBrative nucleoside transporter suBtypes of Ehrlich cells and to assess the contriBution of Na+-dependent concentrative transport mechanisms to the cellular accumulation of nucleoside analogues By these cells. <B>FormycinB> B is poorly metaBolized By mammalian cells and, hence, can Be used as a suBstrate to measure transport kinetics in energetically competent cells. Initial studies estaBlished that <B>FormycinB> B inhiBited [3H]uridine uptake By the ei (equiliBrative inhiBitor-insensitive) and es (equiliBrative inhiBitor-sensitive) transporters of Ehrlich cells with Ki values of 48 +/- 28 and 277 +/- 25 microM, respectively. Similarly, [3H]<B>FormycinB> B had Km values of 111 +/- 52 and 635 +/- 147 microM for uptake By the ei and es transporters, respectively. When assays were conducted in the presence of Na+, plus 100 nM nitroBenzylthioinosine to prevent efflux via the es transporters, the intracellular concentration of [3H]<B>FormycinB> B exceeded the initial medium concentration By more than 3-fold, indicating the activity of a Na+-dependent transporter. Interestingly, the initial rate of uptake of [3H]<B>FormycinB> B was significantly higher in the Li+ Buffer (es-mediated Vmax = 65 +/- 10 pmol/microliter . sec) than in the Na+ Buffer (Vmax = 8.4 +/- 0.9 pmol/microliter . sec); this may reflect trans-acceleration of [3H]<B>FormycinB> B uptake By elevated intracellular adenosine levels resulting from the low Na+ environment. This model was then used to assess the interaction of gemcitaBine (2',2'-difluorodeoxycytidine) with the equiliBrative and concentrative nucleoside transporters. GemcitaBine, which has shown consideraBle potential for the treatment of solid tumors, was a relatively poor inhiBitor of [3H]<B>FormycinB> B uptake via the equiliBrative transporters (IC50 approximately 400 microM). In contrast, gemcitaBine was a potent inhiBitor of the Na+-dependent nucleoside transporter of Ehrlich cells (IC50 = 17 +/- 5 nM). These results suggest that the cellular expression/activity of Na+-dependent nucleoside transporters may Be an important determinant in gemcitaBine cytotoxicity and clinical efficacy.
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Effect of cellular differentiation on nucleoside transport in human neuroBlastoma cells.
Brain Research, 1994Co-Authors: Kenneth W Jones, R. Jane Rylett, James R. HammondAbstract:ABstract The nucleoside transport characteristics of undifferentiated and differentiated LA-N-2 human neuroBlastoma cells were compared through measurement of the cellular accumulation of [3H]<B>FormycinB> B in the aBsence and presence of specific nucleoside transport Blockers such as dipyridamole and nitroBenzylthioinosine (NBMPR). [3H]NBMPR was also used as a high affinity proBe to oBtain an estimate of the numBer of NBMPR-sensitive nucleoside transport proteins. Undifferentiated LA-N-2 cells accumulated [3H]<B>FormycinB> B (25 μM) via a NBMPR/ dipyridamole sensitive, Na+-independent, nucleoside transport system (Vi = 1.52 pmol/μl/s; maximum intracellular concentration = 45 pmol/μl cell water). The undifferentiated cells also had a high density of site-specific [3H]NBMPR Binding sites (135,000 sites/cell; KD= 0.4 nM). When cell differentiation was induced By exposure to a serum-free defined medium, the initial rate of transporter-mediated [3H]<B>FormycinB> B uptake increased to 1.92 pmol/μl/s, and the steady-state intracellular concentration of [3H]<B>FormycinB> B also increased significantly to 73 pmol/gml. However, there was no concominant change in the numBer of [3H]NBMPR Binding sites, and the additional uptake was not Na+-dependent. This enhanced uptake in the differentiated cells appeared to Be due, in part, to an increased functional expression of a NBMPR-resistant form of facilitated nucleoside transporter. Approximately 18% of the transporter-mediated uptake in the differentiated cells was resistant to inhiBition By NBMPR at concentrations that Blocked transport completely in the undifferentiated cells. This cell model may prove useful for Basic studies on regulation of nucleoside transporter suBtype expression in neural tissues, and for evaluation of the efficacy and potential host toxicity of cytotoxic nucleoside analogues (± specific transport Blockers) in the treatment of neuroBlastoma.
Kathleen M. Giacomini - One of the best experts on this subject based on the ideXlab platform.
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<B>FormycinB> B Elimination from the CereBrospinal Fluid of the Rat
Pharmaceutical Research, 1993Co-Authors: Andrew C. Hui, Kathleen M. GiacominiAbstract:The goal of this study was to determine whether specific transport systems are involved in nucleoside elimination from the cereBrospinal fluid (CSF). First, in vitro studies were carried out in isolated choroid plexus tissue slices from rat to ascertain the mechanisms of transport of <B>FormycinB> B, a model nucleoside analogue. ^3H-<B>FormycinB> B accumulated against a concentration gradient in the presence of an Na^+ gradient in the isolated ATP-depleted choroid plexus tissue slices. This accumulation was reduced By high concentrations of unlaBeled <B>FormycinB> B. NitroBenzylthioinosine (NBMPR), an equiliBrative nucleoside transport inhiBitor, inhiBited the uptake of <B>FormycinB> B in the aBsence of an Na^+ gradient. These data suggest that Both equiliBrative and secondary active Na^+-nucleoside transport systems are present in rat choroid plexus. In vivo , <B>FormycinB> B, together with inulin as a Bulk flow marker, was injected into the lateral ventricle of the anesthetized rat with the aid of a stereotaxic device, and CSF was sampled from the cisterna magna at various times after injection. Twelve rats were randomized and divided into a low- and a high-dose group. The CSF clearance (CL_CSF) of <B>FormycinB> B was significantly higher than the CL_CSF of inulin in Both animal groups ( P < 0.01), indicating that <B>FormycinB> B is cleared from CSF By a pathway(s) in addition to Bulk flow. <B>FormycinB> B CL_CSF was significantly lower in the high-dose group than in the low-dose group ( P < 0.05), suggesting a saturaBle CSF elimination. The CL_CSF of <B>FormycinB> B was also significantly reduced in animals treated with NBMPR ( P < 0.05). These data are consistent with the in vitro studies and collectively suggest that <B>FormycinB> B is eliminated from the CSF By a pathway(s) in addition to Bulk flow. At least one pathway is saturaBle and may represent an equiliBrative nucleoside transport system which can Be inhiBited By NBMPR.
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Sodium-dependent nucleoside transport in choroid plexus from raBBit. Evidence for a single transporter for purine and pyrimidine nucleosides.
The Journal of biological chemistry, 1992Co-Authors: Gang Yuan, Andrew C. Hui, Claire M. Brett, Kathleen M. GiacominiAbstract:The overall goal of this study was to determine the mechanisms By which nucleosides are transported in choroid plexus. Choroid plexus tissue slices oBtained from raBBit Brain were depleted of ATP with 2,4-dinitrophenol. Uridine and thymidine accumulated in the slices against a concentration gradient in the presence of an inwardly directed Na+ gradient. The Na(+)-driven uptake of uridine and thymidine was saturaBle with Km values of 18.1 +/- 2.0 and 13.0 +/- 2.3 microM and Vmax values of 5.5 +/- 0.3 and 1.0 +/- 0.2 nmol/g/s, respectively. Na(+)-driven uridine uptake was inhiBited By naturally occurring riBo- and deoxyriBonucleosides (adenosine, cytidine, and thymidine) But not By synthetic nucleoside analogs (dideoxyadenosine, dideoxycytidine, cytidine araBinoside, and 3'-azidothymidine). Both purine (guanosine, inosine, <B>FormycinB> B) and pyrimidine nucleosides (uridine and cytidine) were potent inhiBitors of Na(+)-thymidine transport with IC50 values ranging Between 5 and 23 microM. <B>FormycinB> B competitively inhiBited Na(+)-thymidine uptake and thymidine trans-stimulated <B>FormycinB> B uptake. These data suggest that Both purine and pyrimidine nucleosides are suBstrates of the same system. The stoichiometric coupling ratios Between Na+ and the nucleosides, guanosine, uridine, and thymidine, were 1.87 +/- 0.10, 1.99 +/- 0.35, and 2.07 +/- 0.09, respectively. The system differs from Na(+)-nucleoside co-transport systems in other tissues which are generally selective for either purine or pyrimidine nucleosides and which have stoichiometric ratios of 1. This study represents the first direct demonstration of a unique Na(+)-nucleoside co-transport system in choroid plexus.
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Nucleoside transport in Brush Border memBrane vesicles from human kidney.
Biochimica et biophysica acta, 1992Co-Authors: Marcelo M. Gutierrez, Andrew C. Hui, Claire M. Brett, Ronda J. Ott, Kathleen M. GiacominiAbstract:The goal of this study was to elucidate the mechanisms of nucleoside transport in the Brush Border memBrane of the human kidney. [3H]Uridine was transported into Brush Border memBrane vesicles (BBMV) from human kidney via Na(+)-independent and Na(+)-dependent processes. The Na(+)-dependent transport was saturaBle (Km = 4.76 +/- 0.39 microM; Vmax = 6.42 +/- 0.17 pmol/mg proteins per s) and was trans-stimulated By unlaBeled uridine. Structural analogs of uridine (100 microM), 2'-deoxyuridine (2-dU) and dideoxyuridine (ddU), significantly inhiBited Na(+)-uridine uptake into BBMV. Previous studies have suggested that Na(+)-nucleoside co-transport occurs via two major systems (Vijayalakshmi et al. (1988) J. Biol. Chem. 263, 19419-19423). One system (cit) is generally pyrimidine-selective; thymidine serves as a model suBstrate. The other system (cif) is generally purine-selective; <B>FormycinB> B serves as a model suBstrate. Uridine and adenosine are suBstrates of Both systems. Thymidine and cytidine (100 microM), But not <B>FormycinB> B (100 microM) inhiBited Na(+)-uridine uptake. In addition, [3H]thymidine exhiBited an Na(+)-driven overshoot phenomenon whereas [3H]<B>FormycinB> B did not. Na(+)-thymidine uptake was inhiBited By (100 microM) adenosine, uridine, guanosine, But not By <B>FormycinB> B and inosine. Further studies demonstrated that guanosine trans-stimulated thymidine uptake suggesting that guanosine and thymidine share a common transporter in the human renal BBMV. A different pattern was identified in BBMV from the raBBit kidney where Both [3H]thymidine and [3H]<B>FormycinB> B as well as [3H]uridine exhiBited a transient Na(+)-driven overshoot phenomenon. Collectively, these data suggest that in raBBit renal BBMV Both cif and cit systems are present whereas in human renal BBMV, there appears to Be a single concentrative Na(+)-nucleoside cotransport system that interacts with uridine, cytidine, thymidine, adenosine and guanosine But not with <B>FormycinB> B and inosine. The system is similar to the previously descriBed cit system except that guanosine is also a suBstrate.
Leroy B. Townsend - One of the best experts on this subject based on the ideXlab platform.
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The Synthesis of a New Pyrazolo[3,4-c]pyridine C-Nucleoside, StructurallyRelated to <B>FormycinB> B
Synlett, 2002Co-Authors: Vassilios N. Kourafalos, Nicole Pouli, Panagiotis Marakos, Leroy B. TownsendAbstract:The first preparation of the 4-deaza analogue of <B>FormycinB> B is descriBed, via the reaction of 3-acetamido-2-methoxy-4-methylpyridine with a suitaBly protected riBonolactone and suBsequent ring closure to result in the 3-suBstituted pyrazolo[3,4-c]pyridine riBoside 12.
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Nucleosides. 5. Synthesis of guanine and <B>FormycinB> B derivatives as potential inhiBitors of purine nucleoside phosphorylase
Journal of medicinal chemistry, 1993Co-Authors: Ji Wang Chern, Horng Yuh Lee, Chien Shu Chen, Donna S. Shewach, Peter E. Daddona, Leroy B. TownsendAbstract:In an effort to develop potent human purine nucleoside phosphorylase (PNP) inhiBitors as immunosuppressive and chemotherapeutic agents, several 8-aminoguanine derivatives were synthesized and evaluated as potential PNP inhiBitors. These studies were designed to investigate the hydrophoBic effect of a suBstituent on the N-9 of the purine heterocycle and/or the C-5' positions. Compounds such as 8-aminoguanosine, guanosine, <B>FormycinB> B, and 8-aminoacyclovir containing a p-(fluorosulfonyl)Benzoyl moiety were synthesized. The affinity of these compounds to erythrocytic PNP was determined and none of these compounds showed a Better affinity than those of the parent compounds. However, we found that the effect of hydrophoBicity at the N-9 and the C-5' positions might play an important role in Binding to the active site of PNP. Thus, 8-amino-5'-deoxy-5'-(phenylthio)guanosine (19) was found to Be the Best inhiBitor in this series of compounds with a Ki = 0.45 microM.
F E Parkinson - One of the best experts on this subject based on the ideXlab platform.
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Effect of adenosine receptor agonists on release of the nucleoside analogue [3H]<B>FormycinB> B from cultured smooth muscle DDT1 MF-2 cells.
European journal of pharmacology, 1998Co-Authors: S L Borgland, F E ParkinsonAbstract:Adenosine has receptor-mediated effects in a variety of cell types and is predominantly formed from ATP By a series of nucleotidase reactions. Adenosine formed intracellularly can Be released By Bidirectional nucleoside transport processes to activate cell surface receptors. We examined whether stimulation of adenosine receptors has a regulatory effect on transporter-mediated nucleoside release. DDT1 MF-2 smooth muscle cells, which possess nitroBenzylthioinosine-sensitive (ES) transporters as well as Both adenosine A1 and A2 receptors, were loaded with the metaBolically staBle nucleoside analogue [3H]<B>FormycinB> B. N6-cyclohexyladenosine (CHA), a selective adenosine A1 receptor agonist, produced a concentration-dependent inhiBition of [3H]<B>FormycinB> B release with an IC50 value of 2.7 microM. Further investigation revealed CHA interacts directly with nucleoside transporters with a Ki value of 3.3 microM. Neither 5'-N-ethylcarBoxamidoadenosine (NECA), a mixed adenosine A1 and A2 receptor agonist, nor CGS 21680, a selective adenosine A2A receptor agonist, affected nucleoside release. We conclude that release of the nucleoside <B>FormycinB> B from DDT1 MF-2 cells is not regulated By adenosine A1 or A2 receptor activation.
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uptake and release of 3h <B>FormycinB> B via sodium dependent nucleoside transporters in mouse leukemic l1210 ma27 1 cells
Journal of Pharmacology and Experimental Therapeutics, 1997Co-Authors: S L Borgland, F E ParkinsonAbstract:At least seven functionally distinct nucleoside transport processes exist; however, mouse leukemic L1210/MA27.1 cells possess only one suBtype, a Na+-dependent transporter termed N1/ cif . The capacity of this transporter suBtype to release nucleosides from L1210/MA27.1 cells was investigated with the poorly metaBolized inosine analog [3H]<B>FormycinB> B. Uptake of [3H]<B>FormycinB> B into these cells was inhiBited By replacement of Na+ in the Buffer with choline, or By Blocking Na+/K+ ATPase with 2 mM ouaBain, inhiBiting glycolysis with 5 mM iodoacetic acid or inhiBiting nucleoside transport with 1 mM phloridzin. Sodium stimulated uptake with an EC50 value of 12 mM. To measure release of [3H]<B>FormycinB> B, cells were loaded with [3H]<B>FormycinB> B (10 μM) then washed and resuspended in Buffer. Replacement of Na+ in the Buffer with choline enhanced [3H]<B>FormycinB> B release By 20 to 47%, and significant stimulation of release was oBserved with Na+concentrations of 30 mM or less. Resuspending loaded cells into Na+ Buffer containing 2 mM ouaBain or 10 μM monensin, a Na+ ionophore, significantly enhanced [3H]<B>FormycinB> B release during 20 min By 39% or 29%, respectively. Release of [3H]<B>FormycinB> B into choline Buffer was inhiBited 26.5% By 10 mM phloridzin and 39.6% By 10 mM propentofylline, compounds known to inhiBit various transporters including Na+-dependent nucleoside transporters. Release was also inhiBited significantly By 100 μM concentrations of dilazep, dipyridamole and nitroBenzylthioinosine, inhiBitors with selectivity for Na+-independent nucleoside transporters. In the aBsence of Na+, the permeants adenosine and uridine enhanced [3H]<B>FormycinB> B release By up to 40.9% and 21.4%, respectively. These data indicate that in the aBsence of an inwardly directed Na+ gradient, Na+-dependent nucleoside transporters can function in the release of nucleosides.
Agnieszka Bzowska - One of the best experts on this subject based on the ideXlab platform.
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interactions of calf spleen purine nucleoside phosphorylase with <B>FormycinB> B and its aglycone spectroscopic and kinetic studies
Nucleosides Nucleotides & Nucleic Acids, 2007Co-Authors: Jacek Wierzchowski, Beata Iwańska, Agnieszka Bzowska, David ShugarAbstract:Phosphorolysis of 7-methylguanosine By calf spleen purine nucleoside phosphorylase (PNP) is weakly inhiBited, uncompetitively, By <B>FormycinB> B (FB) with K i = 100 μ M and more effectively By its aglycone (7KPP), IC50 35–100 μ M. In striking contrast, 7KPP inhiBits the reverse reaction (synthesis of 8-azaguanosine from 8-azaguanine) competitively, with K i ∼ 2–4 μ M. <B>FormycinB> B forms only a weakly fluorescent complex with PNP, and 7KPP even less so, indicating that Both ligands Bind as the neutral, not anionic, forms. 7KPP is a rare example of a PNP non-suBstrate inhiBitor of Both the phosphorolytic and reverse synthetic pathways.
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Interactions of Calf Spleen Purine Nucleoside Phosphorylase with <B>FormycinB> B and its Aglycone—Spectroscopic and Kinetic Studies
Nucleosides nucleotides & nucleic acids, 2007Co-Authors: Jacek Wierzchowski, Beata Iwańska, Agnieszka Bzowska, David ShugarAbstract:Phosphorolysis of 7-methylguanosine By calf spleen purine nucleoside phosphorylase (PNP) is weakly inhiBited, uncompetitively, By <B>FormycinB> B (FB) with K i = 100 μ M and more effectively By its aglycone (7KPP), IC50 35–100 μ M. In striking contrast, 7KPP inhiBits the reverse reaction (synthesis of 8-azaguanosine from 8-azaguanine) competitively, with K i ∼ 2–4 μ M. <B>FormycinB> B forms only a weakly fluorescent complex with PNP, and 7KPP even less so, indicating that Both ligands Bind as the neutral, not anionic, forms. 7KPP is a rare example of a PNP non-suBstrate inhiBitor of Both the phosphorolytic and reverse synthetic pathways.
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Crystal structure of the ternary complex of E. coli purine nucleoside phosphorylase with <B>FormycinB> B, a structural analogue of the suBstrate inosine, and phosphate (Sulphate) at 2.1 A resolution.
Journal of molecular biology, 1998Co-Authors: Gertraud Koellner, David Shugar, Marija Luić, Wolfram Saenger, Agnieszka BzowskaAbstract:ABstract The ternary complex of purine nucleoside phosphorylase from E. coli with <B>FormycinB> B and a sulphate or phosphate ion crystallized in the hexagonal space group P6122 with unit cell dimensions a = 123.11, c = 241.22 A and three monomers per asymmetric unit. The Biologically active hexamer is formed through 2-fold crystallographic symmetry, constituting a trimer of dimers. High-resolution X-ray diffraction data were collected using synchrotron radiation (DaresBury, England). The crystal structure was determined By molecular replacement and refined at 2.1 A resolution to an R-value of 0.196. There is one active centre per monomer, composed of residues Belonging to two suBunits of one dimer. The phosphate Binding site is strongly positively charged and consists of three arginine residues (Arg24, Arg87 and Arg43 from a neighBouring suBunit), Ser90 and Gly20. It is occupied By a sulphate or phosphate anion, each oxygen atom of which accepts at least two hydrogen Bonds or salt-Bridges. The sulphate or phosphate anion is also in direct contact with the riBose moiety of <B>FormycinB> B. The riBose Binding site is composed of Ser90, Met180, Glu181 and His4, the latter Belonging to the neighBouring suBunit. The Base Binding site is exposed to solvent, and the Base is unspecifically Bound through a chain of water molecules and aromatic-aromatic interactions. In all monomers the nucleosides are in the high syn conformation aBout the glycosidic Bonds with χ in the range 100 to 130°. The architecture of the active centre is in line with the known Broad specificity and the kinetic properties of E. coli PNP.