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The reactions of a ribonuclease model substrate, the compound uridine-3'-p-nitrophenyl phosphate, have been examined using heavy-atom isotope effects and stereochemical analysis. The cyclization of this compound is subject to catalysis by general base (by imidazole buffer), specific base (by carbonate buffer), and by acid. All three reactions proceed by the same mechanistic sequence, via cyclization to cUMP, which is stable under basic conditions but which is rapidly hydrolyzed to a mixture of 2'- and 3'-UMP under acid conditions. The isotope effects indicate that the specific base-catalyzed reaction exhibits an earlier transition state with respect to bond cleavage to the leaving group compared to the general base-catalyzed reaction. Stereochemical analysis indicates that both of the base-catalyzed reactions proceed with the same stereochemical outcome. It is concluded that the difference in the nucleophile in the two base-catalyzed reactions results in a difference in the transition state structure but both reactions are most likely concerted, with no phosphorane intermediate. The (15)N isotope effects were also measured for the reaction of the substrate with ribonuclease A. The results indicate that considerably less negative charge develops on the leaving group in the transition state than for the general base-catalyzed reaction in solution. Copyright 2000 Academic Press. 相似文献
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硫酯酶(thioesterase, TE)具有区域定向性(regiospecific)、化学定向性(chemospecific)及立体定向性(stereospecific)的特点。这些特性决定了TE作为生物催化剂(biocatalysis)在工业生产中具有较高的应用价值和广阔的应用前景。McyC-TE (microcystin thioesterase, McyC TE)来自铜绿微囊藻(microcystis aeruginosa)NRPS/PKS生物合成基因簇。我们利用正交试验提高McyC TE表达量,得到稳定的诱导表达条件,并结合成熟的线性多肽化学合成法对其底物适用性做了进一步研究。得到的最佳诱导表达条件为:诱导时机2 h,诱导剂异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside, IPTG)浓度0.75 mmol/L,诱导时间6 h,诱导转速210 r/min,诱导温度20 ℃,使TE的表达量由8.75 mg/L提高至22.15 mg/L,时间缩短了6.5 h。TE表达量的大幅度提升和表达时间的缩短为将来酶的结构及催化机制研究奠定了基础。TE底物适用性研究结果发现:McyC TE并不遵循“4 n + 2原则”;底物中转角过多不仅不利于环肽的形成,更可能形成卷曲影响环化;无D型氨基酸亦可通过加入其它位阻较小较灵活的Gly或者自带天然转角Pro的可弱化肽链的刚性,促进催化反应;含苯环的Phe的引入在一定程度上阻碍了环化;底物无肽链氨基酸数目奇偶性的选择;延长多肽链长度也可环化,McyC-TE的底物容忍度较大,使天然多肽药物筛选范围增大,也为增强天然多肽药物药效增加了改良方案,为进一步研究McyC TE的催化功能提供了实验基础。 相似文献
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Sakuzo Fukui 《Bioscience, biotechnology, and biochemistry》2013,77(2):321-324
The relation between the rate of increase in nonprotein nitrogenous compounds (NPN) of rabbit muscle and muscle pH ranging from 5.9 to 7.2 was examined during the post-mortem storage. Muscle of a high ultimate pH was prepared by the injection of ICH2COOH into the vein. The more the muscle pH kept away from 6.3, the more NPN increased. Therefore, it has been suggested that the post-mortem proteolysis is mainly attributed to the acid proteolytic system comprising cathepsins in muscles at a pH lower than 6.3 and to the neutral proteolytic system in muscles at a pH higher than 6.3.The ratio of the increment of ninhydrin positive materials to that of Cu-Folin phenol reagent positive materials among NPN was relatively large in muscles at a high pH. This result has suggested that the neutral proteolytic system was more abound in exopeptidase activity than acid proteolytic system. 相似文献
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Preparation of Uridine Diphosphate-N-Acetylgalactosamine from Uridine Diphosphate-N-Acetylglucosamine by Using Microbial Enzymes
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A method was developed for the large scale preparation of uridine diphosphate-N-acetylgalactosamine (UDP-GalNAc) from uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) by means of microbial enzymes. With Bacillus subtilis cell-free extract as a source of UDP-GlcNAc 4-epimerase, about 35% of the UDP-GlcNAc added was converted to UDP-GalNAc. After the residual UDP-GlcNAc was degraded to uridine triphosphate and N-acetylglucosamine-1-phosphate with a protamine-treated extract of bakers' yeast as a source of UDP-GlcNAc pyrophosphorylase, UDP-GalNAc was separated by anion-exchange column chromatography. The nucleotide was recovered by adsorption on charcoal and elution with ammoniacal ethanol. The final yield was about 100 μmol. 相似文献
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Girish B. Chheda Henry A. Tworek Arvind K. Bhargava Elliot Rachlin Shib P. Dutta Helen B. Patrzyc 《Nucleosides, nucleotides & nucleic acids》2013,32(4):417-429
Abstract A new modified nucleoside, 3-(3-amino-3-carboxypropyl)-uridine was isolated from a 24 hour collection of a normal human urine. The structure was assigned on the basis of UV, NMR and mass spectrometry data and confirmed by comparison of the spectral data and HPLC mobilities with those of an authentic sample. Origin and significance of this nucleoside in relation to tRNA is discussed. The new nucleoside is present also in the urine of cancer patients but in smaller amounts. 相似文献
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Valery Andrushchenko Daniele Padula Elena Zhivotova Shigeki Yamamoto Petr Bouř 《Chirality》2014,26(10):655-662
Lanthanide complexes exhibit interesting spectroscopic properties yielding many applications as imaging probes, natural chirality amplifiers, and therapeutic agents. However, many properties are not fully understood yet. Therefore, we applied magnetic circular dichroism (MCD) spectroscopy, which provides enhanced information about the underlying electronic structure to a series of lanthanide compounds. The metals in the M3+ state included Y, La, Eu, Tb, Dy, Ho, Er, Tm, Yb, and Lu; the spectra were collected for selected tetraphenylporphin (TPP) and octaethylporphin (OEP) complexes in chloroform. While the MCD and UV‐VIS absorption spectra were dominated by the porphyrin signal, metal binding significantly modulated them. MCD spectroscopy was found to be better suited to discriminate between various species than absorption spectroscopy alone. The main features and trends in the lanthanide series observed in MCD and absorption spectra of the complexes could be interpreted at the Density Functional Theory (DFT) level, with effective core potentials on metal nuclei. The sum over state (SOS) method was used for simulation of the MCD intensities. The combination of the spectroscopy and quantum‐chemical computations is important for understanding the interactions of the metals with the organic compounds. Chirality 26:655–662, 2014. © 2014 Wiley Periodicals, Inc. 相似文献
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M. Draminski A. Zgit-wroblewska A. Drabikowska D. Shugar 《Nucleosides, nucleotides & nucleic acids》2013,32(1-2)
Abstract Various acyclonucleoside analogues have been examined as inhibitors of highly purified, and electrophoretically homogeneous, bacterial uridine phosphorylase. 相似文献
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NIEMAN R. H.; CLARK R. A.; PAP D.; OGATA G.; MAAS E. V. 《Journal of experimental botany》1988,39(3):301-309
Nieman, R. H., Clark, R. A., Pap, D., Ogata, G. and Maas, E.V. 1988. Effects of salt stress on adenine and uridine nucleotidepools, sugar and acid-soluble phosphate in shoots of pepperand safflower.-J. exp. Bot. 39: 301309. Pepper (Capsicum annuum cv. Yolo wonder) and safflower (Carthamustinctonus L. cv. Gila) were grown hydroponically and subjectedto a salt stress (51 mol m3 NaCl plus 25.5 mol m3CaCl2). Mature photosynthetic source leaves and shoot meristematicsinks (young pepper leaves and safflower buds) were analyzedfor nucleotides by high performance liquid chromatography andfor hexose and acid-soluble Ppepper was still vegetativewhereas safflower had switched to flower bud formationthesalt stress reduced the fresh shoot yield of pepper by nearlytwo-thirds and of safflower by half. It reduced the ATP pooland ATP/ADP ratio in the source leaves of both species and alsoin the young pepper leaves. It had little or no effect on ATPor other nucleotide pools in safflower buds. The UDPG pool wasnot affected in source leaves or safflower buds, but in theyoung pepper leaves it was reduced by half, along with UTP.These reductions were accompanied by over a 3-fold increasein hexose and a large decrease in ester phosphate. In safflower,on the other hand, salt stress had little or no effect on UDPG,hexose, or ester phosphate in either source leaves or buds.The results suggest that salt stress reduced the growth of pepperbecause it reduced assimilation of photosynthate, possibly aconsequence of reduced UDPG, UTP, and ATP pools in the growingleaves. Salt stress did not so markedly affect assimilationof photosynthate in the more tolerant safflower. Key words: Growth suppression, energy charge, UDPG 相似文献
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In the serum-deficient medium, the cultured Swiss 3T3 and CHO-K1 cells transit to the resting state. The rates of uridine phosphorylation and RNA synthesis in these cells are lowered. After the addition of fresh medium containing 10% serum, cell proliferation is induced. At the early stage of cell entrance into the cell cycle uridine transport through the cell plasma membrane remains unchanged in both cultures. During the 1st hour after serum addition the rate of uridine phosphorylation increases in 3T3 cells to remain practically unchanged in CHO-K1 cells. At this time, RNA synthesis in cells increases almost twofold in both cultures. A correlation has been revealed between the initial level of uridine phosphorylation in 3T3 cells and the percentage of its maximal elevation after serum addition. No such a correlation was observed for CHO-K1 cells. The rate of uridine phosphorylation in arrested CHO-K1 cells is higher than that in 3T3 cells. It has been included that the initial increase of uridine phosphorylation during serum stimulation may be not obligatory for all cell types, but depends on the level of uridine kinase activity before serum addition to the cells. 相似文献
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Uridine diphosphate-D-glucose dehydrogenase of hen oviduct 总被引:5,自引:0,他引:5
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Maurycy Szlenkier Karol Kamel Jerzy Boryski 《Nucleosides, nucleotides & nucleic acids》2016,35(8):410-425
Mitsunobu reaction of partially acylated uridine proceeds with high regioselectivity for intramolecular SN2 anhydro linkage closuring. Under the reaction conditions, an isomeric mixture of diacyl uridine derivatives with either free 2′- or 3′-hydroxyl group was transformed into a single cyclonucleosidic product, 2,2′-anhydro-3′,5′-di-O-acyluridine. This paper presents a possible mechanism of the reactions, the explanation of observed phenomenon based on semiempirical and density functional theory (DFT) calculations and possible utility of this synthetic pathway. 相似文献
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Uridine transport and phosphorylation were studied in cultured Swiss 3T3 CHO-K1 cells, differing in their growth characteristics. Uridine was shown to be transported to the cell with a high rate. With the 2 micronM uridine concentration in the medium, the stationary level of free uridine in cells is reached 10 seconds following incubation at 25 degrees, and the further uridine uptake is limited by phosphorylation.. The uridine transport to the cell does not depend on the DNA synthesis level and the growth phase of 3T3 and CHO-K1 cells. With the increase in culture density, the rate of uridine phosphorylation decreases in 3T3 cells being actually unchanged in CHO-K1 cells. With the equal cell density in both the cases, the phosphorylation rate in CHO-K1 cells is by several times higher than that in 3T3 cells. A positive correlation between uridine phosphorylation rate and DNA synthesis has been observed under various cultivation condition of CHO-K1 cells. 相似文献
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