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PubMed1991

Production of 5-methyluridine by immobilized thermostable purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus JTS 859.

5-Methyluridine was produced continuously from thymine and inosine by immobilized enzymes, which consisted of thermostable purine nucleoside phosphorylase and thermostable pyrimidine nucleoside phosphorylase obtained from Bacillus stearothermophilus JTS 859. The process was carried out in a column reactor at 60 degrees C for 17 d without any bacterial contamination under non-aseptical conditions. Half-lives of the activity of the immobilized enzymes were 47 d and 4.5 d at 60 degrees C and 70 degrees C, respectively, although half-life of the crude enzyme was only 14 h at 70 degrees C.

Bacterial Proteins↗

PubMed2006

The role of thymidine phosphorylase and uridine phosphorylase in (fluoro)pyrimidine metabolism in peripheral blood mononuclear cells.

Thymidine phosphorylase (TP) and uridine phosphorylase (UP) catalyze the (in)activation of several fluoropyrimidines, depending on their catalytic activity and substrate specificity. Blood cells are the first compartment exposed to most anticancer agents. The role of white blood cells in causing toxic side effects and catalyzing drug metabolism is generally underestimated. Therefore we determined the contribution of the white blood cell compartment to drug metabolism, and we investigated the activity and substrate specificity of TP and UP for the (fluoro)pyrimidines thymidine (dThd), uridine (Urd), 5'-deoxy-5-fluorouridine (5' dFUrd) and 5-fluorouracil (5FU) in peripheral blood mononuclear cells (PBMC) and undifferentiated monocytes and differentiated monocytes: macrophages and dendritic cells. PBMC had an IC50 of 742 microM exposed to 5'dFUrd, increasing to > 2000 microM when both TP and UP activities were inhibited. Total phosphorolytic activity was higher with dThd than with Urd, 5'dFUrd or 5FU. Using a specific TP inhibitor (TPI) and UP inhibitor (BAU) we concluded that dThd and Urd were preferentially converted by TP and UP, respectively, while 5'dFUrd and 5FU were mainly converted by TP (about 80%) into 5FU and FUrd, respectively. 5FU was effectively incorporated into RNA. dThd conversion into thymine was highest in dendritic cells (52.6 nmol thymine/h/10(6) cells), followed by macrophages (two-fold) and undifferentiated monocytes (eight-fold). TPI prevented dThd conversion almost completely. In conclusion, PBMC were relatively insensitive to 5'dFUrd, and the natural substrates dThd and Urd were preferentially converted by TP and UP, respectively. TP and UP were both responsible for converting 5'dFUrd/5FU into 5FU/FUrd, respectively.

Cells, Cultured↗

PubMed2004

Synthesis and evaluation of 6-methylene-bridged uracil derivatives. Part 2: optimization of inhibitors of human thymidine phosphorylase and their selectivity with uridine phosphorylase.

A series of novel 6-methylene-bridged uracil derivatives have been optimized for clinical use as the inhibitors of human thymidine phosphorylase (TP). We describe their synthesis and evaluation. Introduction of a guanidino or an amidino group enhanced the in vitro inhibitory activity of TP comparing with formerly reported inhibitor 1. Their selectivity for TP based on uridine phosphorylase inhibitory activity was also evaluated. Compound 2 (TPI) has been selected for clinical evaluation based on its strong TP inhibition and excellent modulation of 2'-deoxy-5-(trifluoromethyl)uridine (F(3)dThd) pharmacokinetics. As a result, TAS-102 (a combination of F(3)dThd and TPI) is currently in phase 1 clinical studies.

Absorption↗

PubMed2002

Induction of thymidine phosphorylase by interferon and taxanes occurs only in human cancer cells with low thymidine phosphorylase activity.

Thymidine phosphorylase (TP) regulates intracellular thymidine metabolism. It has been reported to be a prognostic factor for tumor angiogenesis and to activate some prodrugs of 5-fluorouracil (5-FU) to 5-FU. There is also evidence that TP is induced by interferons (IFNs) and xenobiotics, such as cyclophosphamide and taxanes, in experimental human cancer cells and xenografts. We investigated the induction of TP expression by IFNalpha and Paclitaxel in vitro and in vivo in human tumor cells with low and with high TP activity. TP activity in KB, NUGC-3, and KOC2S cells, which had low TP activity, was increased 2 to 4 fold by IFNalpha, but was still lower than in non-treated SHIN-3 and HRA cells, which have high TP activity. IFNalpha did not promote TP activity in SHIN-3 and HRA cells, but expression of TP mRNA increased 2 to 4 fold in response to IFNalpha in all cells tested. These results suggest that the expression of TP protein would be regulated post-transcriptionally by another factor after IFN-induced amplification of TP mRNA. A single dose of Paclitaxel to nude mice xenografted with KB and KM20C tumors, expressing low TP activity, increased TP activity about 4 to 7 fold compared to non-treated tumors. In contrast, TP expression in MX-1 and H-31 tumors was originally high and did not change by the treatment of Paclitaxel. The activities of uridine phosphorylase in all tumors used showed no changes in response to IFNalpha or Paclitaxel. We determined the level of STAT1alpha, an IFN-inducible transcription factor of the TP gene, and found that it was low in low TP expressing tumor cells and markedly increased to about 4 fold by IFN, almost reaching the level in high TP expressing cells whose STAT1alpha level was unchanged by IFN. When TP activity and STAT1alpha expression in clinically resected colorectal cancers were simultaneously measured, almost all tumors had high expression of both TP and STAT1alpha. In conclusion, our results suggest that IFN and Paclitaxel affect human cancer cells with low TP activity but not those with high TP activity and that the STAT1alpha expression may reflect TP activity, at least in experimental human cancer cells.

Animals↗

PubMed1996

Divalent cations but not other activators enhance phosphorylase kinase's affinity for glycogen phosphorylase.

To better understand the physical interaction between glycogen phosphorylase-b (P-b) and its only known kinase, phosphorylase kinase (PbK) and the relationship of this interaction to the activation of PbK, direct binding studies are necessary. By utilizing an enzyme-linked immunosorbent assay, a method was developed for measuring the binding of PbK to immobilized P-b under a variety of experimental conditions. A monoclonal antibody specific for the alpha subunit of PbK that had no effect on the phosphorylation of P-b by PbK or on the interaction of PbK with known effectors was used to detect PbK bound to plated P-b. Hyperbolic binding curves were obtained regardless of whether the concentration of Pbk or P-b was varied, and the assay detected changes in relative affinity caused by certain effectors of the kinase. The allosteric effector ADP, alkaline pH, and phosphorylation by cAMP-dependent protein kinase, all activators of PbK, did not cause significant changes in its relative affinity for P-b; however, Ca2+ and Mg2+ ions, which also stimulate PbK, increased its affinity for P-b, with Mg2+ being more effective. Mn2+, which inhibits the P-b conversion activity of PbK, was found to be the most potent enhancer of its affinity for P-b, although divalent cations may enhance binding. Inclusion of ATP analogs in the binding assay with Ca2+ and Mg2+ to stimulate catalytic assay conditions did not further affect the apparent affinity for P-b, which is consistent with the previously reported rapid equilibrium random bi-bi kinetic mechanism for P-b conversion.

Animals↗

PubMed1984

Allosteric interactions of glycogen phosphorylase b. A crystallographic study of glucose 6-phosphate and inorganic phosphate binding to di-imidate-cross-linked phosphorylase b.

The binding to glycogen phosphorylase b of glucose 6-phosphate and inorganic phosphate (respectively allosteric inhibitor and substrate/activator of the enzyme) were studied in the crystal at 0.3 nm (3A) resolution. Glucose 6-phosphate binds in the alpha-configuration at a site that is close to the AMP allosteric effector site at the subunit-subunit interface and promotes several conformational changes. The phosphate-binding site of the enzyme for glucose 6-phosphate involves contacts to two cationic residues, Arg-309 and Lys-247. This site is also occupied in the inorganic-phosphate-binding studies and is therefore identified as a high-affinity phosphate-binding site. It is distinct from the weaker phosphate-binding site of the enzyme for AMP, which is 0.27 nm (2.7A) away. The glucose moiety of glucose 6-phosphate and the adenosine moiety of AMP do not overlap. The results provide a structural explanation for the kinetic observations that glucose 6-phosphate inhibition of AMP activation of phosphorylase b is partially competitive and highly co-operative. The results suggest that the transmission of allosteric conformational changes involves an increase in affinity at phosphate-binding sites and relative movements of alpha-helices. In order to study glucose 6-phosphate and phosphate binding it was necessary to cross-link the crystals. The use of dimethyl malondi-imidate as a new cross-linking reagent in protein crystallography is discussed.

Adenosine Monophosphate↗

PubMed1976

Hybrid enzyme of liver phosphorylase and phosphorylase I.

A hybrid enzyme (LI) of liver phosphorylase [EC 2.4.1.1.] (L) and phosphorylase I, which is mainly located in brain, was isolated and its enzymatic and immunological properties were examined. The following results were obtained: (1) AMP stimulated the b forms of the hybrid (LIb), I(Ib), and L(Lb); (2) in the presence of AMP, SO42- stimulated Lb more than LIb and inhibited Ib; (3) in the absence of AMP, SO42- stimulated all three isozymes in the order: Ibeta less than LI less than Lbeta; (4) on conversion to the a forms, the activities of L, LI, and I increased 35.5-fold, 3-fold, and 1.2-fold, respectively; (5) the relative inhibition potencies of anti-Lb antibody with LIa and LIb were 63% and 4%, respectively of that with La, and those of anti-Ib antibody with LIa and LIb were 42% and 88%, respectively of that with Ia. Since the ratios of the specific activities of purified La and Ia and of Lbeta and Ibeta are 70: 82 and 2 : 70, respectively (Schliselfeld, 1973), the present findings suggest a 1 : 1 association of I and L subunits in the hybrid molecule.

Adenosine Monophosphate↗

PubMed2002

Targeted deletion of both thymidine phosphorylase and uridine phosphorylase and consequent disorders in mice.

Thymidine phosphorylase (TP) regulates intracellular and plasma thymidine levels. TP deficiency is hypothesized to (i) increase levels of thymidine in plasma, (ii) lead to mitochondrial DNA alterations, and (iii) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In order to elucidate the physiological roles of TP, we generated mice deficient in the TP gene. Although TP activity in the liver was inhibited in these mice, it was fully maintained in the small intestine. Murine uridine phosphorylase (UP), unlike human UP, cleaves thymidine, as well as uridine. We therefore generated TP-UP double-knockout (TP(-/-) UP(-/-)) mice. TP activities were inhibited in TP(-/-) UP(-/-) mice, and the level of thymidine in the plasma of TP(-/-) UP(-/-) mice was higher than for TP(-/-) mice. Unexpectedly, we could not observe alterations of mitochondrial DNA or pathological changes in the muscles of the TP(-/-) UP(-/-) mice, even when these mice were fed thymidine for 7 months. However, we did find hyperintense lesions on magnetic resonance T(2) maps in the brain and axonal edema by electron microscopic study of the brain in TP(-/-) UP(-/-) mice. These findings suggested that the inhibition of TP activity caused the elevation of pyrimidine levels in plasma and consequent axonal swelling in the brains of mice. Since lesions in the brain do not appear to be due to mitochondrial alterations and pathological changes in the muscle were not found, this model will provide further insights into the causes of MNGIE.

Animals↗

PubMed1985

Comparative study of the interactions AMP-phosphorylase b and AMP analogues-phosphorylase b.

The different effects induced by AMP and its analogues on the tertiary structure and the coenzyme environment of phosphorylase b were studied by titration of the slowly reacting thiol groups and by quenching of the coenzyme fluorescence, respectively, to determine the possible differences that activate phosphorylase b. The following results were obtained: The coenzyme environment depends upon the nucleotide studied. AMP, when bound to its first site, opens the coenzyme pocket. The slow cysteines were shielded by the nucleotides against their DTNB titration depending on the nucleotide studied. The enzyme difference spectra in presence of the nucleotide showed that the negative band of 260 nm is similar for all nucleotides possessing the same base, but the positive band obtained in the presence of AMP was diminished when other nucleotides were present.

Adenosine Monophosphate↗

PubMed1995

Molecular cloning and nucleotide sequence of purine nucleoside phosphorylase and uridine phosphorylase genes from Klebsiella sp.

Klebsiella sp. LF 1202 was isolated as a bacterium that can assimilate adenosine as a sole source of carbon and nitrogen [F. Ling et al., Agric. Biol. Chem., 55, 573-575 (1991)] from a soil sample. Both the purine nucleoside phosphorylase (PNPase) and uridine phosphorylase (UPase) of this bacterium were induced simultaneously when the bacterium was cultured in a medium containing adenosine or uridine as a sole source of carbon and nitrogen. This induction profile is different from that of Escherichia coli. Here we cloned and sequenced the gene corresponding to each enzyme. The open reading frame (ORF) of the PNPase gene consisted of 717 bp that encoded a polypeptide of 239 amino acids with a molecular weight of 26,198. The ORF of the UPase gene consisted of 834 bp that encoded a polypeptide of 278 amino acids with a molecular weight of 28,912.

Amino Acid Sequence↗

PubMed1996

Purification and characterization of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus TH 6-2.

The purine nucleoside phosphorylase (Pu-NPase) and the pyrimidine nucleoside phosphorylase (Py-NPase) have been purified from Bacillus stearothermophilus TH 6-2. The Pu-NPase is a trimer of 30-kDa subunits and the Py-NPase is a dimer of 46-kDa subunits. The isoelectric points of Pu-NPase and Py-NPase were pH 4.3 and 4.6, respectively. The Pu-NPase could catalyze the phosphorolysis of inosine and guanosine, but not adenosine. the Py-NPase could phosphorolyze both uridine and thymidine.

Amino Acid Sequence↗

PubMed1992

Control of phosphorylase b conformation by a modified cofactor: crystallographic studies on R-state glycogen phosphorylase reconstituted with pyridoxal 5'-diphosphate.

Previous crystallographic studies on glycogen phosphorylase have described the different conformational states of the protein (T and R) that represent the allosteric transition and have shown how the properties of the 5'-phosphate group of the cofactor pyridoxal phosphate are influenced by these conformational states. The present work reports a study on glycogen phosphorylase b (GPb) complexed with a modified cofactor, pyridoxal 5'-diphosphate (PLPP), in place of the natural cofactor. Solution studies (Withers, S.G., Madsen, N.B., & Sykes, B.D., 1982, Biochemistry 21, 6716-6722) have shown that PLPP promotes R-state properties of the enzyme indicating that the cofactor can influence the conformational state of the protein. GPb complexed with pyridoxal 5'-diphosphate (PLPP) has been crystallized in the presence of IMP and ammonium sulfate in the monoclinic R-state crystal form and the structure refined from X-ray data to 2.8 A resolution to a crystallographic R value of 0.21. The global tertiary and quaternary structure in the vicinity of the Ser 14 and the IMP sites are nearly identical to those observed for the R-state GPb-AMP complex. At the catalytic site the second phosphate of PLPP is accommodated with essentially no change in structure from the R-state structure and is involved in interactions with the side chains of two lysine residues (Lys 568 and Lys 574) and the main chain nitrogen of Arg 569. Superposition of the T-state structure shows that were the PLPP to be incorporated into the T-state structure there would be a close contact with the 280s loop (residues 282-285) that would encourage the T to R allosteric transition. The second phosphate of the PLPP occupies a site that is distinct from other dianionic binding sites that have been observed for glucose-1-phosphate and sulfate (in the R state) and for heptulose-2-phosphate (in the T state). The results indicate mobility in the dianion recognition site, and the precise position is dependent on other linkages to the dianion. In the modified cofactor the second phosphate site is constrained by the covalent link to the first phosphate of PLPP. The observed position in the crystal suggests that it is too far from the substrate site to represent a site for catalysis.

Amino Acid Sequence↗

PubMed1976

Study on the structure-function relationship of polynucleotide phosphorylase: model of a proteolytic degraded polynucleotide phosphorylase.

It is already known that modification of E. coli polynucleotide phosphorylase by endogenous proteolysis induces drastic changes in both phosphorolysis and polymerisation reactions. The structural parameters of the proteolysed polynucleotide phosphorylase are described. The phosphorolysis of polynucleotide, which is quite progressive for the native enzyme, is shown to be only partially progressive for the degraded enzyme, owing to the loss of polymer attachment sites.

Escherichia coli↗

PubMed2002

Cloning of the maltose phosphorylase gene from Bacillus sp. strain RK-1 and efficient production of the cloned gene and the trehalose phosphorylase gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis.

The maltose phosphorylase (MPase) gene of Bacillus sp. strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme. The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1. This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask. About 60% of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B. subtilis.

Amino Acid Sequence↗

PubMed1999

A mutation in GLUT2, not in phosphorylase kinase subunits, in hepato-renal glycogenosis with Fanconi syndrome and low phosphorylase kinase activity.

Fanconi-Bickel syndrome is characterized by hepato-renal glycogenosis with severe renal tubular dysfunction and rickets. It has recently been found to be associated with GLUT2 mutations in three families. In another family, low activities of liver phosphorylase kinase (Phk) have been observed, suggesting that Fanconi-Bickel syndrome might be genetically heterogeneous. We have analyzed this family for mutations in the GLUT2 gene and in the three Phk subunit genes that can cause liver glycogenosis (PHKA2, PHKB, and PHKG2). The coding sequences of all three Phk genes are normal but we have identified a homozygous missense mutation (Pro417Leu) in GLUT2. The affected proline residue is completely conserved in all mammalian glucose permease isoforms and even in bacterial sugar transporters and is believed to be critical for the passage of glucose through the permease. Seven affected individuals from different branches of the same large consanguineous sibship all are homozygous for this mutation. These findings indicate that there is no specific subtype of genetic Phk deficiency giving rise to hepato-renal glycogenosis. Rather, they provide further evidence that Fanconi-Bickel syndrome is caused by GLUT2 mutations. The low Phk activity is probably a secondary phenomenon that contributes to the deposition of glycogen in response to the intracellular glucose retention caused by GLUT2 deficiency.

Amino Acid Sequence↗

PubMed1986

Function of pyridoxal 5'-phosphate in glycogen phosphorylase: 19F NMR and kinetic studies of phosphorylase reconstituted with 6-fluoropyridoxal and 6-fluoropyridoxal phosphate.

19F NMR spectroscopic properties of glycogen phosphorylase reconstituted with 6-fluoropyridoxal (6-FPAL) and 6-fluoropyridoxal phosphate (6-FPLP) were investigated. Analysis of the contribution of chemical shift anisotropy to the line width of the 6-FPLP-enzyme signal shows that the coenzyme molecule is tightly bound to the protein. The chemical shift of the fluorine nucleus in the free 6-FPLP protein is pH independent from pH 6 to pH 9.1. When the 6-FPLP-enzyme forms complexes with AMP, AMP plus glucose-1-P, and AMP plus inorganic phosphate, signals at -11.0, -13.1, and -10.4 ppm are observed, respectively. These different chemical shifts indicate that the protein in each complex has a distinct conformation. The exchange rate between the 6-FPLP-protein-AMP complex and the same complex with bound glucose-1-P is estimated to be 3300 +/- 700 s-1, and that between the 6-FPLP-protein-AMP complex and with bound inorganic phosphate is 500 +/- 100 s-1. The former exchange rate is 13 times faster than that of the same process for the 6-FPAL-enzyme. Analysis of the effects of temperature on the 19F line shape of the 6-FPLP enzyme in the presence of ligands shows that the exchange rates between different complexes drop significantly between 20 and 10 degrees C. Within this temperature range, Arrhenius plots of the enzymatic activities of the native and 6-FPLP-enzymes at varied temperatures also show a pronounced curvature.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗

PubMed1992

Inhibitors of human purine nucleoside phosphorylase. Synthesis of pyrrolo[3,2-d]pyrimidines, a new class of purine nucleoside phosphorylase inhibitors as potentially T-cell selective immunosuppressive agents. Description of 2,6-diamino-3,5-dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3,2-d] pyrimidin-4-one.

Purine nucleoside phosphorylase (PNP) is a purine-metabolizing enzyme in the purine cascade and has been a target for drug design for sometime. A series of potent human PNP inhibitors, pyrrolo[3,2-d]pyrimidines (9-deazaguanines), has been synthesized and evaluated in the enzyme assay and in the cell line assay using MOLT-4 (T-cell) and MGL-8 (B-cell) lymphoblasts for selectivity. One of the compounds, 2,6-diamino-3,5- dihydro-7-(3-thienylmethyl)-4H-pyrrolo[3,2-d]pyrimidine-4-one (11c; CI-972), was found to be moderately potent, competitive, and reversible inhibitor of PNP with Ki = 0.83 microM. It was also found to be selectively cytotoxic to MOLT-4 lymphoblasts (IC50 = 3.0 microM) but not to MGL-8 lymphoblasts and was evaluated further. Compound 11c (CI-972) is under development in the clinic.

B-Lymphocytes↗

PubMed1986

Inhibitors of human purine nucleoside phosphorylase. Synthesis, purine nucleoside phosphorylase inhibition, and T-cell cytotoxicity of 2,5-diaminothiazolo[5,4-d]pyrimidin-7(6H)-one and 2,5-diaminothiazolo[4,5-d]pyrimidin-7(6H)-one. Two thio isosteres of 8-aminoguanine.

8-Aminoguanine is a potent inhibitor of purine nucleoside phosphorylase (PNP) and also a substrate of PNP. Two thio isosteres of 8-aminoguanine, 2,5-diaminothiazolo[5,4-d]pyrimidin-7(6H)-one (2) and 2,4-diaminothiazolo[4,5-d]pyrimidin-7(6H)-one (3), which cannot be substrates of PNP, were synthesized and evaluated for their inhibitory activity against PNP. They were found to be weak inhibitors of PNP and to be noncytotoxic for MOLT-4 T-cells in culture.

Cell Division↗