Purification and Characterization of Thermostable Purine Nucleoside Phosphorylase of Bacillus stearothermophilus JTS 859

A thermostable purine nucleoside phosphorylase has been purified more than 800-fold from Bacillus stearothermophilus JTS 859. The enzyme had a molecular weight of 68,000 consisting of 2 identical subunits (A/_w, 34,000). The isoelectric point of the enzyme was 4.7. The enzyme did not contain cysteine. The optimal pH of the enzyme reaction was from 7.5 to 11.0. The Michaelis constants for inosine, guanosine, 2′-deoxyinosine, and 2′-deoxyguanosine were 0.22, 0.14, 0.20, and 0.10mM, respectively. The optimal temperature of the reaction was 80 C. The half-life of the enzyme was 16 hr in 20mM potassium phosphate and ImM inosine (pH 7.0) at 80°C, and no decrease of the enzyme activity was observed at least for the first 30 hr at 70°C.     

Purine Nucleoside Phosphorylase Activity in Rat Cerebrospinal Fluid

The sequential hydrolysis of purines is present in rat CSF and generates nucleosides as inosine and guanosine that are usual substrates for purine nucleoside phosphorylase (PNP). PNP catalyzes phosphorolysis of the purine nucleosides and deoxynucleosides releasing purine bases. Here we investigated the presence of PNP in CSF of rats using: i) a specific chromophoric analogue of nucleosides, 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG), and ii) an inhibitor of PNP activity, immucillin-H. Additionally, we performed a preliminary kinetic characterization (K(M): Henry-Michaelis-Menten constant; V: maximal velocity) for MESG and inorganic phosphate (Pi). The values of K(M) and V for MESG (n = 3, mean+/-SD) were 142.5+/-29.5 microM and 0.0102+/-0.0006 U mg(-1), respectively. For Pi (n=3, mean+/-SD), the K(M) values and V were 186.8+/-43.7 microM and 0.0104+/-0.0016 U mg(-1), respectively. The results indicated that PNP is present in rat CSF and provided a preliminary kinetic characterization.     

枯草芽孢杆菌TM903嘌呤核苷磷酸化酶的纯化及酶学性质研究

目的：对枯草芽孢杆菌TM903嘌呤核苷磷酸化酶进行分离纯化及酶学性质研究。方法：经加热、硫酸铵盐析和SephadexG-100凝胶过滤,对枯草芽孢杆菌TM903中的嘌呤核苷磷酸化酶进行分离纯化,并对其酶学性质进行研究。结果：酶的最适反应温度为65℃,最适反应pH值为7．5,在30-50℃时热稳定性较好;K^＋对该酶有激活作用,而Na^＋、ca^＋、Mg^＋、Mn^＋等金属离子对该酶有抑制作用;Km值为2．11mmol／L,Vmax值为0．84mmol／（min·L）。结论：分离纯化了枯草芽孢杆菌TM903嘌呤核苷磷酸化酶,并研究了其酶学性质,为利巴韦林的发酵工艺优化提供了重要的酶学理论基础。     

Inhibition of Purine Nucleoside Phosphorylase by Phosphonoalkylpurines 1

Abstract

Optimum inhibition of human erythrocyte purine nucleoside phosphorylase by 9-(phosphonoalkyl)hypoxanthines required an alkyl chain of five carbons or longer. Appropriate modifications of either the base or phosphonate side chain resulted in increased inhibitory activity.     

Properties of Purine Nucleoside Phosphorylase from Enterobacter cloacae

Purine nucleoside phosphorylase from Enterobacter cloacae KY3074 was partially purified by ammonium sulfate fractionation, column chromatography on DEAE-cellulose and DEAE-Sephadex A-50, and gel filtration on Sephadex G-100 and Sepharose 4B. The molecular weight of the enzyme was calculated to be about 87,000 by a gel filtration method on Sephadex G-200. The enzyme was found to be most active at pH 7.5 to 8.5 and 50°C, stable between pH 7.0 and 7.3, and the activity was nearly lost above 70°C. The enzyme split 2´-deoxyinosine and ribonucleosides. Lineweaver-Burk plots for phosphate were non-linear, showing substrate activation. The break-down of inosine approached an equilibrium when approximately 14% of inosine was phosphorylated.     

Purine Nucleoside Phosphorylase Deficiency: A Mutation Update

Purine nucleoside phosphorylase (PNPase) deficiency is an autosomal recessive disorder affecting purine degradation and salvage pathways. Clinically, patients typically present with severe immunodeficiency, neurological dysfunction, and autoimmunity. Biochemically, PNPase deficiency may be suspected in the presence of hypouricemia. We report biochemical and genetic data on a cohort of seven patients from six families identified as PNPase deficient. In all patients, inosine, deoxyinosine, guanosine, and deoxyguanosine were elevated in urine, and mutation analysis revealed seven different mutations of which three were novel. The mutation c.770A>G resulted in the substitution p.His257Arg. A second novel mutation c.257A>G (p.His86Arg) was identified in two siblings and a third novel mutation, c.199C>T (p.Arg67X), was found in a 2-year-old female with delayed motor milestones and recurrent respiratory infections. A review of the literature identified 67 cases of PNPase deficiency from 49 families, including the cases from our own laboratory. PNPase deficiency was confirmed in 30 patients by genotyping and 24 disease causing mutations, including the three novel mutations described in this paper, have been reported to date. In five of the seven patients, plasma uric acid was found to be within the pediatric normal range, suggesting that PNPase deficiency should not be ruled out in the absence of hypouricemia.     

Inhibition and Structure of Toxoplasma gondii Purine Nucleoside Phosphorylase

The intracellular pathogen Toxoplasma gondii is a purine auxotroph that relies on purine salvage for proliferation. We have optimized T. gondii purine nucleoside phosphorylase (TgPNP) stability and crystallized TgPNP with phosphate and immucillin-H, a transition-state analogue that has high affinity for the enzyme. Immucillin-H bound to TgPNP with a dissociation constant of 370 pM, the highest affinity of 11 immucillins selected to probe the catalytic site. The specificity for transition-state analogues indicated an early dissociative transition state for TgPNP. Compared to Plasmodium falciparum PNP, large substituents surrounding the 5′-hydroxyl group of inhibitors demonstrate reduced capacity for TgPNP inhibition. Catalytic discrimination against large 5′ groups is consistent with the inability of TgPNP to catalyze the phosphorolysis of 5′-methylthioinosine to hypoxanthine. In contrast to mammalian PNP, the 2′-hydroxyl group is crucial for inhibitor binding in the catalytic site of TgPNP. This first crystal structure of TgPNP describes the basis for discrimination against 5′-methylthioinosine and similarly 5′-hydroxy-substituted immucillins; structural differences reflect the unique adaptations of purine salvage pathways of Apicomplexa.     

Substrate/Inhibitor Properties of Tumour Purine Nucleoside Phosphorylase

Abstract

Substrate/inhibitor properties of purine nucleoside phosphorylase (PNP), isolated from human lung and kidney tumour tissues, have been characterised and compared with those of the enzyme from the corresponding normal organs.     

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.     

Gene Therapy of Cancer: Activation of Nucleoside Prodrugs with E. coli Purine Nucleoside Phosphorylase

Abstract

During the last few years, many gene therapy strategies have been developed for various disease targets. The development of anticancer gene therapy strategies to selectively generate cytotoxic nucleoside or nucleotide analogs is an attractive goal. One such approach involves the delivery of herpes simplex virus thymidine kinase followed by the acyclic nucleoside analog ganciclovir. We have developed another gene therapy methodology for the treatment of cancer that has several significant attributes. Specifically, our approach involves the delivery of E. coli purine nucleoside phosphorylase, followed by treatment with a relatively non-toxic nucleoside prodrug that is cleaved by the enzyme to a toxic compound. This presentation describes the concept, details our search for suitable prodrugs, and summarizes the current biological data.     

Interactions of Calf Spleen Purine Nucleoside Phosphorylase with Antiviral Acyclic Nucleoside Phosphonate Inhibitors: Kinetics and Emission Studies

Abstract

Association between calf spleen purine nucleoside phosphorylase and a series of phosphonylalkoxyalkyl derivatives of purine bases was studied by inhibition kinetics and fluorimetric titrations. Dissociation constants, determined by fluorimetric titration in phosphate-free conditions, were lower than inhibition constants in 1 mM phosphate, and inhibition was still weaker in 50 mM phosphate, in accord with the postulated bisubstrate analogue character of this class of inhibitors.     

Cloning,Expression, Purification,and Some Properties of Calf Purine Nucleoside Phosphorylase

Calf spleen purine nucleoside phosphorylase (PNP) is considered a model enzyme for the trimeric PNPs subfamily. PCR amplification of the calf phosphorylase from the calf spleen library, cloning, overexpression of the recombinant PNP, its enzymatic activity and interactions with typical ligands of mammalian wild type PNP are described. Relative activity of the recombinant phosphorylase versus several substrates is similar to the respective values obtained for the enzyme isolated from calf spleen. As for the nonrecombinant calf PNP, the unusual fluorescence properties of the PNP/guanine complex were observed and characterized.     

Production of Bacteriophage by Temperature-Sensitive Sporulation Mutants of Bacillus cereus T

Five temperature-sensitive sporulation mutants of Bacillus cereus T have been isolated. These mutants are blocked at stage 0 of sporulation at the restrictive temperature (37 C) but are able to sporulate at nearly normal frequencies at the permissive temperature (26 C). A bacteriophage that forms a stable lysogen in the parent strain is induced at increased frequencies in the mutants. This induction is accompanied, in some of the mutants, by a reduction in immunity to the phage. Revertants, selected for their ability to sporulate normally at both temperatures, lose their ability to produce high titers of the phage. In addition to this lytic phage, an apparently defective phage has been found in lysates of the mutants. Strains cured of the plaque-forming phage still carry the defective phage. Comparisons of physical and biological properties of the plaque-forming phage with those of the two Bacillus cereus phages most similar to it have shown that this phage is not identical to either of them. The maximal titer of phage produced in cultures of the parent strain is about 10(3) plaque-forming units (PFU) per ml at both temperatures. The maximal titers of phage produced by the mutant are 4 x 10(9) PFU/ml at 37 C and 7 x 10(8) PFU/ml at 26 C. Both mutant and parent strains release over 90% of the phage they produce after the onset of stationary phase.     

Purine Nucleoside Phosphorylase Targeted by Annexin V to Breast Cancer Vasculature for Enzyme Prodrug Therapy

Background and Purpose

The targeting of therapeutics is a promising approach for the development of new cancer treatments that seek to reduce the devastating side effects caused by the systemic administration of current drugs. This study evaluates a fusion protein developed as an enzyme prodrug therapy targeted to the tumor vasculature. Cytotoxicity would be localized to the site of the tumor using a protein fusion of purine nucleoside phosphorylase (PNP) and annexin V. Annexin V acts as the tumor-targeting component of the fusion protein as it has been shown to bind to phosphatidylserine expressed externally on cancer cells and the endothelial cells of the tumor vasculature, but not normal vascular endothelial cells. The enzymatic component of the fusion, PNP, converts the FDA-approved cancer therapeutic, fludarabine, into a more cytotoxic form. The purpose of this study is to determine if this system has a good potential as a targeted therapy for breast cancer.

Methods

A fusion of E. coli purine nucleoside phosphorylase and human annexin V was produced in E. coli and purified. Using human breast cancer cell lines MCF-7 and MDA-MB-231 and non-confluent human endothelial cells grown in vitro, the binding strength of the fusion protein and the cytotoxicity of the enzyme prodrug system were determined. Endothelial cells that are not confluent expose phosphatidylserine and therefore mimic the tumor vasculature.

Results

The purified recombinant fusion protein had good enzymatic activity and strong binding to the three cell lines. There was significant cell killing (p<0.001) by the enzyme prodrug treatment for all three cell lines, with greater than 80% cytotoxicity obtained after 6 days of treatment.

Conclusion

These results suggest that this treatment could be useful as a targeted therapy for breast cancer.     

Sporulation of a Cortexless Mutant of a Variant of Bacillus cereus

A stage 4 sporulation mutant of a strain of Bacillus cereus var. alesti fails to synthesize a cortex although all other structural components appear normal. With terminal lysis the spore core as well as the sporangium is lysed. Both the uptake of (45)Ca and the synthesis of dipicolinic acid (DPA) are similar to these activities in the parent strain, but these components (DPA and Ca) are lost to the medium with the drastic lysis. The first stage of diaminopimelic acid incorporation, that into germ cell wall mucopeptide, is intact in the mutant; the second stage, that into cortical mucopeptide, is absent. These biochemical studies as well as phospholipid metabolism and freeze-etch analysis suggest the lesion lies in the outer forespore membrane.     

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。     

Air-Liquid Interface Biofilms of Bacillus cereus: Formation, Sporulation, and Dispersion

Biofilm formation by Bacillus cereus was assessed using 56 strains of B. cereus, including the two sequenced strains, ATCC 14579 and ATCC 10987. Biofilm production in microtiter plates was found to be strongly dependent on incubation time, temperature, and medium, as well as the strain used, with some strains showing biofilm formation within 24 h and subsequent dispersion within the next 24 h. A selection of strains was used for quantitative analysis of biofilm formation on stainless steel coupons. Thick biofilms of B. cereus developed at the air-liquid interface, while the amount of biofilm formed was much lower in submerged systems. This suggests that B. cereus biofilms may develop particularly in industrial storage and piping systems that are partly filled during operation or where residual liquid has remained after a production cycle. Moreover, depending on the strain and culture conditions, spores constituted up to 90% of the total biofilm counts. This indicates that B. cereus biofilms can act as a nidus for spore formation and subsequently can release their spores into food production environments.     

Possible Involvement of β-Lactamase in Sporulation in Bacillus cereus

Nonreverting beta-lactamase-negative strains were isolated from the beta-lactamase-constitutive strain, Bacillus cereus 569 H. These strains differed from both beta-lactamase-inducible and -constitutive strains not only in failure to produce beta-lactamase but also in failure to autolyze on aging, delayed sporulation, and failure to release free spores from sporangia when produced. The addition of B. cereus beta-lactamase of 15% purity to a final concentration of 10 IU/ml stimulates sporulation and particularly the release of free spores in culture from sporangia of strain 569 (inducible wild-type), 569/H (constitutive mutant of 569), and HPen(-), a nonreverting beta-lactamase strain isolated from 569/H in this laboratory. Cultures of HPen(-) did not release free spores without this treatment. Similar stimulation of sporulation and spore release by beta-lactamase from B. cereus were observed in another beta-lactamase-negative strain derived from 569/H as well as in certain sporogeny mutants of B. subtilis. The beta-lactamase preparation used in these experiments was free of peptidases, proteases, and autolysins capable of solubilizing wall from vegetative cells. These results, taken with our previous finding that a soluble peptidoglycan inducer becomes available in cultures of B. cereus only at sporulation and that normal derepression of beta-lactamase accompanies normal sporulation, suggest that beta-lactamase in B. cereus may be involved in peptidoglycan metabolism during sporulation and possibly the breakdown of sporangial wall with the concomitant release of mature spores.