The Role of Platelet-Derived Endothelial Cell Growth Factor/Thymidine Phosphorylase in Tumor Behavior

Platelet-derived endothelial cell growth-factor (PD-ECGF) is similar to the pyrimidine enzyme thymidine phosphorylase (TP). A high TP expression at tumor sites is correlated with tumor growth, induction of angiogenesis, and metastasis. Therefore, high TP is most likely associated with a poor prognosis. TP is not only expressed in tumor cells but also in tumor surrounding tissues, such as tumor infiltrating macrophages. TP catalyzes the conversion of thymidine to thymine and doxyribose-1-phosphate (dR-1-P). The latter in its parent form or in its sugar form, deoxyribose (dR) may play a role in the induction of angiogenesis. It may modulate cellular energy metabolism or be a substrate in a chemical reaction generating reactive oxygen species. L-deoxyribose (L-dR) and thymidine phosphorylase inhibitor (TPI) can reverse these effects. The mechanism of TP induction is not yet completely clear, but TNF, IL10 and other cytokines have been clearly shown to induce its expression. The various complex interactions of TP give it an essential role in cellular functioning and, hence, it is an ideal target in cancer therapy.     

Upregulation of Platelet Derived Endothelial Cell Growth Factor/Thymidine Phosphorylase by Interferon Alpha

Thymidine phosphorylase (TP) catalyzes the phosphorolytic cleavage of thymidine to thymine and deoxyribose‐1‐phosphate. TP, which is overexpressed in a wide variety of solid tumors, is involved in the activation and inactivation of fluoropyrimidines. TP is known to be regulated by several cytokines and interferons. In our HT29 cell line the TP mRNA and activity expression increased 2–3 fold after treatment with interferon alpha.     

Determination of Thymidine Phosphorylase Activity in Human Blood Cells and Fibroblasts by a Nonradiochemical Assay Using Reversed-Phase High-Performance Liquid Chromatography

In this study, we demonstrated that the highest activity of thymidine phosphorylase (TP) was found in peripheral blood mononuclear (PBM) cells followed by that of thrombocytes and granulocytes whereas no activity of TP could be detected in erythrocytes. The activity of TP in leukocytes proved to be intermediate compared to the TP activity observed in PBM cells and granulocytes. The activity of TP also was readily detectable in human fibroblasts.     

A Semiempirical Transition State Structure for the First Step in the Alkaline Hydrolysis of Cocaine. Comparison between the Transition State Structure,the Phosphonate Monoester Transition State Analog,and a Newly Designed Thiophosphonate Transition State Analog

Semiempirical molecular orbital calculations have been performed for the first step in the alkaline hydrolysis of the neutral benzoylester of cocaine. Successes, failures, and limitations of these calculations are reviewed. A PM3 calculated transition state structure is compared with the PM3 calculated structure for the hapten used to induce catalytic antibodies for the hydrolysis of cocaine. Implications of these calculations for the computer–aided design of transition state analogs for the induction of catalytic antibodies are discussed.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089460020062     

The Effects of Organic Solvent on the Ribosyl Transfer Reaction by Thermostable Purine Nucleoside Phosphorylase and Pyrimidine Nucleoside Phosphorylase from Bacillus stearothermophilus JTS 859

The effects of organic solvents on the reaction rate and equilibrium of the ribosyl transfer reaction catalyzed by thermostable purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus JTS 859 were examined at 60°C. The reaction rate in the presence of 10% acetone was 1.6 times higher than that of the control. Acetone was the best organic solvent among those tested for accelerating the reaction rate without denaturing the enzymes. On the other hand, the reaction rate in the presence of 5% ethyl acetate was 1.5 times higher than that of the control. However the enzymes were denatured completely after 1 h incubation. Consequently, the acceleration was not attributed to the stabilization of the enzymes. The equilibrium constants of the reaction were not influenced by the presence of acetone, methyl or ethyl alcohols.     

The Effects of Organic Solvent on the Ribosyl Transfer Reaction by Thermostable Purine Nucleoside Phosphorylase and Pyrimidine Nucleoside Phosphorylase from Bacillus stearothermophilus JTS 859

The effects of organic solvents on the reaction rate and equilibrium of the ribosyl transfer reaction catalyzed by thermostable purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus JTS 859 were examined at 60°C. The reaction rate in the presence of 10% acetone was 1.6 times higher than that of the control. Acetone was the best organic solvent among those tested for accelerating the reaction rate without denaturing the enzymes. On the other hand, the reaction rate in the presence of 5% ethyl acetate was 1.5 times higher than that of the control. However the enzymes were denatured completely after 1 h incubation. Consequently, the acceleration was not attributed to the stabilization of the enzymes. The equilibrium constants of the reaction were not influenced by the presence of acetone, methyl or ethyl alcohols.     

Thymidine phosphorylase inhibits the expression of proapoptotic protein BNIP3

An angiogenic factor, thymidine phosphorylase (TP), confers resistance to apoptosis induced by hypoxia. We investigated the molecular basis for the suppressive effect of TP on hypoxia-induced apoptosis using Jurkat cells transfected with TP cDNA, Jurkat/TP, and a mock transfectant, Jurkat/CV. TP and 2-deoxy-d-ribose, a degradation product of thymidine generated by TP enzymatic activity, suppressed hypoxia-induced apoptosis. They also inhibited the upregulation of hypoxia-inducible factor (HIF) 1α and the proapoptotic factor, BNIP3, and caspase 3 activation induced by hypoxia. Introduction of siRNA against BNIP3 in Jurkat cells decreased the proportion of apoptotic cells under hypoxic condition. These findings suggest that the suppression of BNIP3 expression by TP prevents, at least in part, hypoxia-induced apoptosis. Expression levels of TP are elevated in many malignant solid tumors and thus 2-deoxy-d-ribose generated by TP in these tumors might play an important role in tumor progression by preventing hypoxia-induced apoptosis.     

合成利巴韦林的关键酶瞟呤核苷磷酸化酶的原核表达及活性分析

目的：在枯草芽孢杆菌中表达嘌呤核苷磷酸化酶（PNPase）并分析其活性。方法：将PNPase的编码基因deoD克隆入pDG148表达载体,构建原核穿梭型表达载体pDG148-deoD,采用电转化方法将表达载体转入枯草芽孢杆菌WB600后诱导表达重组PNPase;研究重组PNPase的活性。结果与结论：获得的重组PNPase活性较对照提高了193．9％,其最适催化条件为65℃、pH7．5、500μmol／L底物浓度和1％1,2,4-三氮唑-3-羧甲酰胺;对重组菌的发酵条件进行了初步优化,IPTG诱导6h后在发酵液中添加0．5％的Tween-80能大幅度提高重组PNPase的酶活力。     

Occurrence of 5''-Deoxy-5''-Methylthioadenosine Phosphorylase in the Mammalian CNS: Distribution and Kinetic Studies on the Rat Brain Enzyme

5'-Deoxy-5'-methylthioadenosine (MTA) phosphorylase catalyzes the cleavage of MTA, a secondary product of polyamine biosynthesis, to 5-methylthioribose-1-phosphate and adenine. The occurrence and the general properties of the enzyme were studied in mammalian brain with the following results. (1) Cerebral tissues contained levels of MTA phosphorylase that were comparable to those occurring in other mammalian tissues. (2) Interspecies differences in the enzyme distribution were quite limited, with the highest specific activity values observed in pig brain. Moreover, the enzyme seemed to be generally more concentrated in the cerebellar fractions. (3) Rat brain MTA phosphorylase was highly localized in the cellular soluble fraction. In the first days of rat life, its specific activity in the whole brain was observed to decline significantly from a value of 17.6 units/mg at 1-5 days of age to 13.7 units/mg at 6-10 days of age, remaining then fairly constant up to maturity. (4) Kinetic studies performed with the soluble enzyme extracted from rat brain showed: a pH optimum of 7.4; a Km value for MTA of about 10 microM; an inhibitory effect of the MTA analog 5'-deoxy-5'-isobutylthioadenosine; and a remarkable resistance of the enzyme to heat treatment.     

Thymidine phosphorylase suppresses Fas-induced apoptotic signal transduction independent of its enzymatic activity

Thymidine phosphorylase (TP) has chemotactic and angiogenic activities resulting from its enzymatic activity in vitro, and it also promotes tumor growth and inhibits apoptosis in vivo. Recently, we have reported that TP plays an important role in Fas-induced apoptosis. Caspase-8 cleavage, subsequent cytochrome c release, and caspase-3 cleavage were prevented in KB cells transfected with a TP cDNA (KB/TP cells). In this study, treatment with thymidine phosphorylase inhibitor (TPI) or thymidine did not affect cell survival of KB/TP cells during Fas-induced apoptosis. Moreover, treatment with thymine or 2-deoxy-D-ribose (degradation products of thymidine generated by TP) also did not affect cell survival of control transfectant (KB/CV) cells during Fas-induced apoptosis. These findings indicate that TP suppresses Fas-induced apoptotic signal transduction independent of its enzymatic activity.     

Mitochondrial Thymidine Kinase 2 but Not Deoxyguanosine Kinase Is Up-Regulated During the Stationary Growth Phase of Cultured Cells

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial phosphorylation of pyrimidine and purine deoxyribonucleosides, and are essential for maintaining mitochondrial dNTP pools for mitochondrial DNA replication. Here the expression of mitochondrial TK2 and dGK in relation to cell growth phases in cultured cells was investigated. TK2 and dGK protein levels in isolated mitochondria and TK2 activity in total cell extracts from U2OS and TK1 deficient L929 cells were determined. We found that TK2 levels were negatively correlated with cell growth rates and there was an exponential increase in TK2 levels in cells entering stationary phase. The expression of dGK did not change and appeared to be constitutive.     

Towards new thymidine phosphorylase/PD-ECGF inhibitors based on the transition state of the enzyme reaction

Computational studies have been conducted to built a closed form of TPase and to characterize the transition state of the phosphorylisis reaction catalyzed by TPase. The results obtained point to a crucial role of His-85 and the O2 of thymine in the catalysis. This modelled transition state forms the basis for the design of new TPase inhibitors.     

反竞争性抑制的非稳态酶动力学布尔函数图解研究

以非稳态酶动力学的布尔函数图形方法,来研究一类反竞争性抑制的非稳态酶动力学问题,推导出此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类反竞争性制酶反应体系的非稳态酶动力学问题。     

竞争性抑制的非稳态酶动力学布尔函数图论研究

以非稳戊酶动力学的布尔函数图形方法,来研究一类竞争性抑制的非稳态酶动力学问题,推导出此类反应的百稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类竞争性抑制酶反应体系的非稳态酶动力学问题。     

一类非竞争性抑制的非稳态酶动力学布尔函数图解研究

本文以非稳态酶动力学的布尔函数图形方法^[1],来研究一类非竞争性抑制的非稳态酶动力学问题,推导出此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类非竞争性抑制的非稳态酶动力学的动力学过程。     

百合鳞茎淀粉磷酸化酶提取条件的优化

为深入研究百合鳞茎淀粉磷酸化酶（SP）的性质及其在鳞茎淀粉代谢中的生理作用,本试验确定了兰州百合鳞茎内SP的最佳提取和测定条件,并对部分酶学性质进行了初步探索。结果表明：SP的最适提取缓冲液为pH5.8的琥珀酸缓冲液;最佳反应体系为以25 mmol·L^-1的Glc-1-P为底物,在30℃条件下反应10 min。在pH5~6范围内,SP活性最高;百合鳞茎的SP不易保存且不耐热,4℃条件下保存12 h,酶活力最高残留50%;40℃条件下保存30 min后,SP活力几乎完全丧失。     

Computational analyses of the conformational itinerary along the reaction pathway of GH94 cellobiose phosphorylase

GH94 cellobiose phosphorylase (CBP) catalyzes the phosphorolysis of cellobiose into α-d-glucose 1-phosphate (G1P) and d-glucose with inversion of anomeric configuration. The complex crystal structure of CBP from Cellvibrio gilvus had previously been determined; glycerol, glucose, and phosphate are bound to subsites −1, +1, and the anion binding site, respectively. We performed computational analyses to elucidate the conformational itinerary along the reaction pathway of this enzyme. autodock was used to dock cellobiose with its glycon glucosyl residue in various conformations and with its aglycon glucosyl residue in the low-energy ⁴C₁ conformer. An oxocarbenium ion-like glucose molecule mimicking the transition state was also docked. Based on the clustering analysis, docked energies, and comparison with the crystallographic ligands, we conclude that the reaction proceeds from ¹S₃ as the pre-transition state conformer (Michaelis complex) via E₃ as the transition state candidate to ⁴C₁ as the G1P product conformer. The predicted reaction pathway of the inverting phosphorylase is similar to that proposed for the first-half glycosylation reaction of retaining cellulases, but is different from those for inverting cellulases. NAMD was used to simulate molecular dynamics of the enzyme. The ¹S₃ pre-transition state conformer is highly stable compared with other conformers, and a conformational change from ⁴C₁ to ^1,4B was observed.     

Random Bi Bi机制的非稳态酶动力学布尔函数图论研究

本文以非稳态酶动力学的布尔函数图形方法^[1],来研究一类Random Bi Bi机制的非稳态酶动力学问题,推导同此类反应的非稳态酶动力学方程,并对此动力学方程进行了讨论,分析了此类Random Bi Bi机制酶反应体系的非稳态酶动力学方程。