产气肠杆菌EAM-Z1尿苷磷酸化酶的分离纯化及性质研究

从产气肠杆菌（Enterobacter aerogenes）突变株EAMZ1中分离出一种具有较高转移酶活性的尿苷磷酸化酶(Upase)。经测定这种Upase的分子量为12.8×104,亚基分子量为4.3×10.4,由３个同型亚基组成。N端氨基酸序列为：MRMVDLIATKRDGGE。等电点为4.46。对尿苷的Km为0.29mmol/L。酶反应的最适pH为7.8,最适温度为50℃。该酶能磷酸化尿苷、胸苷、5氟尿苷、2′脱氧5氟尿苷及尿嘧啶βD阿拉伯呋喃糖,且具有较高的转移酶活性,能将尿苷和5氟尿嘧啶转化成5氟尿苷(一种抗癌药物的中间体),其转化率为47％。该酶的这些特性对于酶法合成核苷类抗肿瘤药物和抗病毒药物是十分有用的。     

林肯链霉菌丙氨酸脱氢酶的纯化和性质

采用硫酸铵分级沉淀、DEAE-纤维素52柱层析、亲和蓝柱层析和琼脂糖凝胶Sepharose6B柱层析的方法,分离纯化了林肯链霉菌丙氨酸脱氢酶,用聚丙烯酰胺凝胶电泳鉴定为单一组分。以凝胶过滤和聚丙烯酰胺梯度凝胶电泳测得该酶的分子量为170000,SDS-聚丙烯酰胺凝胶电泳测得其亚基分子量为42500,表明林肯链霉菌丙氨酸脱氢酶由四个相同的亚基组成。该酶加氨反应最适pH为9．0,脱氨反应最适pH为9．5,加氨反应和脱氨反应的最适温度均为50℃。加氨反应丙氨酸脱氢酶的表现米氏常数km值为：丙酮酸2．08×10－4mol／L,NH4+2．00×10-2mol／L,NADH2．38×10-5mol／L;脱氨反应的Km为：L-Ala1．43×10-2mol／L;NAD+6．67×10-5mol／L。     

假单胞杆菌D-海因酶的纯化及酶学性质

D 海因酶是工业上生产D 型氨基酸的关键酶 ,用热变性 ,硫酸铵沉淀及SepharoseQfastflow ,Phenyl Sepharosefastflow ,Superose 1 2等柱层析步骤从Pseudomonas 2 2 62菌体中分离纯化了该酶 ,纯化倍数约为 60 ,活力回收约为 1 6%。该酶为同源二聚体 ,分子量约为 1 0 9kD ,亚基分子量约为 53 7kD ,反应最适pH为 8 0 ,最适温度为 70℃ ,在pH6.0～ 1 0 0和温度 60℃以下稳定 ,该酶对巯基试剂敏感 ,大多数二价金属离子如镁、锰离子等能促使酶活提高 ,但高浓度锌离子能抑制酶活 ,以二氢尿嘧啶为底物的米氏常数Km =2 .5× 1 0 - 2 mol L。该酶的N末端1 0个氨基酸残基依次为MDKLIKNGTI     

嗜碱菌(Bacillus sp.)ZBAW6的木聚糖酶的分离纯化及其性质

通过硫酸铵分级沉淀 ,阴离子交换层析 ,凝胶过滤 3步从嗜碱菌Bacillussp .ZBAW6纯化了木聚糖酶。结果表明该酶分子量为 4 5kD。N末端序列为DPFAAAVAPL。在pH5 5～ 1 0 5范围内均具有较高酶活性和稳定性 ;最适反应温度为 6 5℃ ,酶活力基本不变。该酶作用于Beech xylan的Km 为 0 1 1mg mL ,Vmax 为 2 3 89μmol (min·mg)。Hg2 + 对该酶有强的抑制作用     

青霉菌m8产半纤维素酶条件及酶性质研究

半纤维素酶可用于造纸工业的生物制浆和废水处理。通过碳、氮源对产胞外半纤维素酶的影响确定了青霉菌m8的适宜培养基 ,即 4 .5 %麦草粉 ,0 .5 0 % (NH4 ) 2 SO4 ,0 .1%KH2 PO4 ,0 .0 5 %MgSO4 ·7H2 O ,0 .0 3%NaCl,0 .33%Tween80 ,0 .15 %CaCO3。在上述培养基中 ,2 8℃恒温振荡 (12 0r min)培养 4 - 6d ,半纤维素酶活力可达 80u ml左右。用DNS法研究了该酶的性质。结果表明 ,其最适pH值为 4 .5 ,最适反应温度为 5 5℃ ;表观Km值为 4 .6× 10 - 2 g L ;该酶的耐热性比较强 ,可被K+ 、Ca2 + 、Mg2 + 离子激活 ,而被Ag+ 、Fe3+ 和Cu2 + 离子抑制。     

林肯链霉菌谷氨酸合酶的纯化和性质

采用硫酸铵分级沉淀、离子交换层析、凝胶过滤和吸附层析等方法 ,分离纯化了林肯链霉菌谷氨酸合酶 ,电泳鉴定为单一组分 .这是链霉菌中的第 1例 .谷氨酸合酶很不稳定 ,向酶缓冲液中加入α -KG ,PMSF ,EDTA ,β 巯基乙醇和甘油可以大大提高其稳定性 .测得全酶分子量为 1 38ku ,亚基分子量为 81和 5 7ku ,表明该酶由2个不相同的亚基构成 .吸收光谱在 380和 440nm附近没有吸收峰 ,表明该酶是不含铁的非黄素蛋白质 .该酶反应的最适 pH为 7 .2 ,最适温度为 30℃ .该酶对NADH ,α KG和L -Gln的表观Km 值分别为 5. 2 1× 1 0 ^-5 ,4. 1 7× 1 0 ^-4 和 4. 35× 1 0 ^-4 mol/L .以NADPH代替NADH作电子供体 ,该酶仍表现出部分活力 .反应产物Glu和NAD⁺,部分金属离子、氨基酸及三羧酸循环中间物对该酶活力有不同程度的抑制作用 .     

地中海诺卡氏菌丙氨酸脱氢酶的纯化和性质

采用硫酸铵分部沉淀、DEAE纤维素-52柱层析和亲和蓝柱层析的方法,分离纯化了地中海诺卡氏菌(Nocardia mediterranei)U-32丙氨酸脱氢酶(ADH),用聚丙烯酰胺凝肢电泳鉴定为单一组份。以聚丙烯酰胺凝胶梯度电泳测得丙氨酸脱氢酶的分子量为228000,SDS-聚丙烯酰胺凝胶电泳测得其亚基分子量为38000,表明地中海诺卡氏菌U-32丙氢脱氢酶由六个相同的亚基组成。ADH加氨反应最适pH为8．5,脱氨反应最适pH为11．5,ADH的pH稳定范围在pH 7．5-11．5。脱氨反应的最适温度为50℃。ADH的米氏常数KM为：丙酮酸4．88×10-4mol／L;NH+44．03×10-3Mol／L;NADH 6．02×10-5Mol／L;L—Ala7．45×10-3mol／L;NAD+6．67×10-5mol／L。 Hill作图法求得ADH的Hill系数n为：ADH对丙酮酸、NADH和NAD+的Hill系数都为1;对L—Ala和NH4+的Hill系数n值分别为0．52和0．51,ADH对L—Ala和NH+4有负协同效应,由此初步推测ADH是一个调节酶。     

N-氨甲酰基-D-氨基酸酰胺水解酶的快速纯化及性质

通过硫酸铵分级沉淀、疏水层析及阴离子交换层析等三步 ,有效地从一菌株NO .2 2 6 2中纯化了N 氨甲酰基 D 氨基酸酰胺水解酶。结果表明 ,酶活性回收约 2 0 %,纯化了 8 4倍。天然PAGE与SDS PAGE分析表明 ,该酶分子为同源四聚体 ,单体分子量约为 3 5kD。酶催化反应的最适pH为 7 7～ 8 0 ,最适温度为 45℃。以N 氨甲酰 DL 丙氨酸为底物时 ,Km =1 3×1 0 - 3 mol L ,Vmax=0 .3 3mol min。二价金属离子Ni2 + 有激活作用 ,Zn2 + 有明显的抑制作用 ,而Co2 + 对酶活无影响。该酶N 末端 8个氨基酸残基依次为TRQKILAF。     

假单胞杆菌D-海因酶的纯化及酶学性质

D-海因酶是工业上生产D-型氨基酸的关键酶,用热变性,硫酸铵沉淀及Sepharose Q fast flow,Phenyl-Sepharose fast flow,Superose 12等柱层析步骤从pseudomonas2262菌体中分离纯化了该酶,纯化倍数约为60,活力回收约为16%.该酶为同源二聚体,分子量约为109kD,亚基分子量约为53.7kD,反应最适pH为8.0,最适温度为70℃,在pH6.0～10.0和温度60℃以下稳定,该酶对巯基试剂敏感,大多数二价金属离子如镁、锰离子等能促使酶活提高,但高浓度锌离子能抑制酶活,以二氢尿嘧啶为底物的米氏常数Km=2.5×10-2mol/L.该酶的N末端10个氨基酸残基依次为MDKLIKNGTI.     

化疗药物对人胃癌SGC-7901细胞系端粒酶活性影响

目的　研究常见化疗药物对人胃癌SGC 790 1细胞系端粒酶活性影响。方法　MTT (噻唑蓝 )法测定化疗药物顺铂、丝裂霉素、阿霉素、 5 氟尿嘧啶、氟铁龙 (脱氧氟尿苷 )对人胃癌SGC 790 1细胞系毒性作用的半数抑制(IC5 0 )浓度 ;用TRAP ELISA法测定 4 0×IC5 0浓度化疗药物作用于SGC 790 1细胞 4h、 2 8h端粒酶活性及IC5 0浓度IC5 0化疗药物作用于SGC 790 1细胞 2 4h、 72h、 12 0h细胞端粒酶活性。结果　高浓度 ,低浓度顺铂、丝裂霉素对SGC 790 1细胞端粒酶活性有完全抑制作用 ;而阿霉素、 5 氟尿嘧啶、氟铁龙对SGC 790 1细胞端粒酶活性有部分抑制作用。结论　阿霉素、 5 氟尿嘧啶、氟铁龙对SGC 790 1细胞端粒酶活性有轻度抑制作用 ;顺铂、丝裂霉素能完全抑制端粒酶活性 ,呈时间浓度效应     

Abnormalities in uridine homeostatic regulation and pyrimidine nucleotide metabolism as a consequence of the deletion of the uridine phosphorylase gene

We report in the present study the critical role of uridine phosphorylase (UPase) in uridine homeostatic regulation and pyrimidine nucleotide metabolism, employing newly developed UPase-/- mice. Our data demonstrate that the abrogation of UPase activity led to greater than a 6-fold increase in uridine concentrations in plasma, a 5-6-fold increase in lung and gut, and a 2-3-fold increase in liver and kidney, as compared with wild type mice. Urine uridine levels increased 24-fold normal in UPase-/- mice. Uridine half-life and the plasma retention of pharmacological doses of uridine were significantly prolonged. Further, in these UPase-/- mice, abnormal uridine metabolism led to disorders of various nucleotide metabolisms. In the liver, gut, kidney, and lung of UPase-/- mice, total uridine ribonucleotide concentrations increased 2-3 times as compared with control mice. Cytidine ribonucleotides and adenosine and guanosine ribonucleotides also increased, although to a lesser extent, in these organs. Most significant deoxyribonucleotide changes were present in the gut and lung of UPase-/- mice. In these tissues, dTTP concentration increased more than 4-fold normal, and dCTP, dGTP, and dATP concentrations rose 1-2 times normal. In kidney, dTTP concentration increased 2-fold normal, and dCTP and dGTP concentrations rose less than 1-fold normal. In addition, the accumulated uridine in plasma and tissues efficiently reduced 5-fluorouracil host toxicity and altered the anesthetic effect of pentobarbital. These data indicate that UPase is a critical enzyme in the regulation of uridine homeostasis and pyrimidine nucleotide metabolism, and 5-fluorouracil activity.     

Properties of Nucleoside Phosphorylase from Enterobacter aerogenes

The properties of uridine Phosphorylase (UPase) and purine nucleoside Phosphorylase (PNPase) at high temperature were investigated. Both enzymes were found to be distributed in a wide range of bacteria and were partially purified from Enterobacter aerogenes AJ 11125 by heat treatment, ammonium sulfate fractionation and column chromatographies onDEAE-cellulose and Sephadex G-150. The UPase was purified 109-fold, and it showed an optimum pH of 8.5 and optimum temperature of 65°C, and activity toward uridine, 2′-deoxyuridine, thymidine and uracil arabinoside but not cytidine. The Km values of UPase for uridine were 0.7 mm at 40°C and 1.8 mm at 60°C. The PNPase was purified 83-fold, and it showed an optimum pH of 6.8 and optimum temperature of 60°C, and significant activity toward purine arabinosides as well as purine ribosides. The Km values of PNPase for inosine were 0.8 mm at 40°C and 2.2 mm at 60°C.     

Biotransformation of the antitumor intermediate 5-fluorouridine by recombinant Escherichia coli with high pyrimidine nucleoside phosporylase activity

Abstract

5-Fluorouridine (5-FUrd) is a precursor of the widely used antitumor drug doxifluridine. We have produced 5-FUrd by biotransformation by cloning the gene encoding pyrimidine nucleoside phosphorylase (PyNPase) from Enterobacter aero-genes CMCC (B) 45103 and expression in Escherichia coli BL21 (DE3), resulting in recombinant E. coli BL21 (DE3)/ pET28a-PyNPase. After medium optimization, the PyNPase activity in the fermentation broth was 1613 U mg^–1, which was 54-fold that of E. aerogenes. Under optimal conditions (cell concentration, 0.5 g L^–1; uridine, 10 mM; 5-fluorouracil, 45 mM; temperature, 50°C; pH, 7.8), more than 90% of uridine was converted to 5-FUrd, suggesting that this is a valuable tool for application in the preparation of antiviral and antitumor drugs.     

Disruption of uridine homeostasis links liver pyrimidine metabolism to lipid accumulation

We report in this study an intrinsic link between pyrimidine metabolism and liver lipid accumulation utilizing a uridine phosphorylase 1 transgenic mouse model UPase1-TG. Hepatic microvesicular steatosis is induced by disruption of uridine homeostasis through transgenic overexpression of UPase1, an enzyme of the pyrimidine catabolism and salvage pathway. Microvesicular steatosis is also induced by the inhibition of dihydroorotate dehydrogenase (DHODH), an enzyme of the de novo pyrimidine biosynthesis pathway. Interestingly, uridine supplementation completely suppresses microvesicular steatosis in both scenarios. The effective concentration (EC₅₀) for uridine to suppress microvesicular steatosis is approximately 20 µM in primary hepatocytes of UPase1-TG mice. We find that uridine does not have any effect on in vitro DHODH enzymatic activity. On the other hand, uridine supplementation alters the liver NAD⁺/NADH and NADP⁺/NADPH ratios and the acetylation profile of metabolic, oxidation-reduction, and antioxidation enzymes. Protein acetylation is emerging as a key regulatory mechanism for cellular metabolism. Therefore, we propose that uridine suppresses fatty liver by modulating the liver protein acetylation profile. Our findings reveal a novel link between uridine homeostasis, pyrimidine metabolism, and liver lipid metabolism.     

Uridine phosphorylase association with vimentin. Intracellular distribution and localization

Uridine phosphorylase (UPase), a key enzyme in the pyrimidine salvage pathway, is associated with the intermediate filament protein vimentin, in NIH 3T3 fibroblasts and colon 26 cells. Affinity chromatography was utilized to purify UPase from colon 26 and NIH 3T3 cells using the uridine phosphorylase inhibitor 5'-amino benzylacyclouridine linked to an agarose matrix. Vimentin copurification with UPase was confirmed using both Western blot analysis and MALDI-MS methods. Separation of cytosolic proteins using gel filtration chromatography yields a high molecular weight complex containing UPase and vimentin. Purified recombinant UPase and recombinant vimentin were shown to bind in vitro with an affinity of 120 pm and a stoichiometry of 1:2. Immunofluorescence techniques confirm that UPase is associated with vimentin in both NIH 3T3 and colon 26 cells and that depolymerization of the microtubule system using nocodazole results in UPase remaining associated with the collapsed intermediate filament, vimentin. Our data demonstrate that UPase is associated with both the soluble and insoluble pools of vimentin. Approximately 60-70% of the total UPase exists in the cytosol as a soluble protein. Sequential extraction of NIH 3T3 or colon 26 cells liberates an additional 30-40% UPase activity associated with a detergent extractable fraction. All pools of UPase have been shown to possess enzymatic activity. We demonstrate for the first time that UPase is associated with vimentin and the existence of an enzymatically active cytoskeleton-associated UPase.     

Effects of 5-fluorouridine on modified nucleosides in mouse liver transfer RNA

Administration of the pyrimidine antimetabolite, 5-fluorouridine, to mice was found to cause a marked specific reduction of the amounts of 5-methyluridine, pseudouridine, and dihydrouridine but not of 3-(3-amino-3-carboxypropyl)uridine in tRNA from the livers of the treated animals. The data presented indicate that this effect is not simply due to the incorporation of 5-fluorouridine into tRNA; the drug appears to interfere directly with the enzymic reactions involved in the modification of the 5-position of uridine. 5-Fluorouridine was found to have no effect on the modification of adenosine, guanosine, and cytidine in mouse liver tRNA.     

Identification of an antilymphocyte transformation substance from Pasteurella multocida

Pasteurella multocida is one of the most important bacteria responsible for diseases of animals. Crude extracts from sonicated P. multocida strain Dainai‐1, which is serotype A isolated from bovine pneumonia, were found to inhibit proliferation of mouse spleen cells stimulated with Con A. The crude extract was purified by cation and anion exchange chromatography and hydroxyapatite chromatography. Its molecular weight was 27 kDa by SDS‐PAGE and it was named PM27. PM27 was found to inhibit proliferation of mouse spleen cells stimulated with Con A as effectively as did the crude extract; however, its activity was lost after heating to 100°C for 20 min. PM27 did not directly inhibit proliferation of HT‐2 cells, which are an IL‐2‐dependent T cell line, nor did it modify IL‐2 production by Con A‐stimulated mouse spleen cells. The N‐terminal amino acid sequence of PM27 was determined and BLAST analysis revealed its identity to uridine phosphorylase (UPase) from P. multocida. UPase gene from P. multocida Dainai‐1 was cloned into expression vector pQE‐60 in Escherichia coli XL‐1 Blue. Recombinant UPase (rUPase) tagged with His at the C‐terminal amino acid was purified with Ni affinity chromatography. rUPase was found to inhibit proliferation of mouse spleen cells stimulated with Con A; however, as was true for PM27, its activity was lost after heating to 100°C for 20 min. Thus, PM27/UPase purified from P. multocida has significant antiproliferative activity against Con A‐stimulated mouse spleen cells and may be a virulence factor.     

Protein engineering of uridine phosphorylase from Escherichia coli K-12. II. Comparative study of hybrid and mutant forms of uridine phosphorylases]

Genes for hybrid uridine phosphorylases (UPases) consisting of fragments of amino acid sequences of UPases from Escherichia coli and Salmonella typhimurium were constructed. Producing strains of the corresponding proteins were genetically engineered. Mutant forms of the E. coli K-12 UPase were produced by site-directed mutagenesis. A comparative study of the enzyme properties of the mutant and hybrid forms of bacterial UPases was performed. It was shown that Asp27 unlike Asp5 and Asp29 residues of the E. coli UPase forms part of the active site of the protein. A scheme of the involvement of Asp27 in the binding of inorganic phosphate is proposed.     

Protein Engineering of Uridine Phosphorylase from Escherichia coliK-12. II. A Comparative Study of the Properties of Hybrid and Mutant Forms of Uridine Phosphorylases

Genes for hybrid uridine phosphorylases (UPases) consisting of fragments of amino acid sequences of UPases from Escherichia coliand Salmonella typhimuriumwere constructed. Producing strains of the corresponding proteins were genetically engineered. Mutant forms of the E. coliK-12 UPase were produced by site-directed mutagenesis. A comparative study of the enzyme properties of the mutant and hybrid forms of bacterial UPases was performed. It was shown that Asp27 rather than Asp5 and Asp29 residues of the E coliUPase forms part of the active site of the protein. A scheme of the involvement of Asp27 in the binding of inorganic phosphate is proposed.