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1.
Most living organisms can synthesize isosinate from 5-phosphoribosyl 1-pyrophosphate in the de novo purine biosynthesis pathway,
which is basically composed of 10 reaction steps. Phosphoribosylglycinamide synthetase (GARS) catalyzes the second step of
the pathway. We found that the enzyme shows weak, but significant, sequence similarity to phosphoribosylglycinamide formyltransferase
2 (GART2) and the ATPase domain of phosphoribosylaminoimidazole carboxylase (AIRCA), which catalyze the third and sixth steps
of the pathway, respectively. In addition, the three enzymes were similar in amino acid sequence to biotin carboxylase (BC)
and carbamoylphosphate synthetase (CPS), which are the members of the GS ADP-forming family. This family has been identified
through a tertiary structure comparison and includes glutathione synthetase, d-alanine:d-alanine ligase, BC, succinyl-CoA synthetase β-chain, and phosphoribosylaminoimidazole-succinocarboxamide synthase. Molecular
phylogenetic analysis based on a multiple alignment of GARS, GART2, AIRCA, BC, and CPS suggests that GART2 is more closely
related to AIRCA than to GARS among the three enzymes from the pathway, though the three enzymes are relatively close to each
other within the GS ADP-forming family. Moreover, the analysis showed that archaeal GARS had diverged before the speciation
between bacteria and eucarya.
Received: 3 June 1998 / Accepted: 8 September 1998 相似文献
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脂肪酶(EC 3.1.1.3)是应用广泛的工业用酶。高效的脂肪酶产生菌是脂肪酶工业生产和应用的前提。通过基因的重新设计与合成技术优化了解脂耶氏酵母(Yarrowia lipolytica)脂肪酶YLL的密码子,并实现了其在毕赤酵母(Pichia pastoris)中的高效表达;通过高通量筛选策略获得了更高效的脂肪酶基因工程菌菌株SILVER。在14 L发酵罐条件下,菌株SILVER酶活达40 500 U/ml、蛋白质含量达2.52 g/L发酵液,为该类脂肪酶的产业化奠定了基础。 相似文献
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6-甲基嘌呤-2'-脱氧核苷(MePdR)是一种新型抗癌药物,它作为药物前体应用于PNP自杀基因治疗系统可以选择性杀伤肿瘤细胞.本实验构建了一个高效表达大肠杆菌来源的嘌呤核苷磷酸化酶重组质粒,并利用基因工程菌以15mmol/L 6-甲基嘌呤和60mmol/L 2'-脱氧尿苷为底物合成6-甲基嘌呤-2'-脱氧核苷,在40mmol/L pH7.0的磷酸缓冲液中,2%菌体在55℃反应2h,转化率可达83.78%.用硅胶制备薄层提纯得到白色针状晶体,收率为76.4%.HPLC测定该产物纯度99.3%,核磁共振鉴定该产物为MePdR. 相似文献
5.
6-甲基嘌呤-2′-脱氧核苷(MePdR)是一种新型抗癌药物,它作为药物前体应用于PNP自杀基因治疗系统可以选择性杀伤肿瘤细胞。本实验构建了一个高效表达大肠杆菌来源的嘌呤核苷磷酸化酶重组质粒,并利用基因工程菌以15mmol/L 6-甲基嘌呤和60mmol/L 2′-脱氧尿苷为底物合成6-甲基嘌呤-2′-脱氧核苷,在40mmol/L pH7.0的磷酸缓冲液中,2%菌体在55℃反应2h,转化率可达83.78%。用硅胶制备薄层提纯得到白色针状晶体,收率为76.4%。HPLC测定该产物纯度99.3%,核磁共振鉴定该产物为MePdR。 相似文献
6.
L. Yu. Sklyarov I. N. Sbitneva N. A. Kopina I. G. Sidorovich 《Russian Journal of Bioorganic Chemistry》2000,26(4):245-256
Pyridoxyl residue was suggested to be used as a multifunctional protective and modifying group in peptide synthesis. The modification
was carried out by introducing the pyridoxyl residue in free or partially protected peptides or by the addition of amino acid
pyridoxyl esters by the methods of conventional peptide synthesis without the removal of the pyridoxyl group at the terminal
stages of the synthesis (the second approach is more convenient). Pyridoxyl residue was also used as a spacer in solid phase
peptide synthesis. It was attached to the polymer by the alkylation of the hydroxyl groups or of the pyridine ring of the
pyridoxyl derivatives with the chloromethylated styrene-divinylbenzene copolymer (the standard Merrifield resin). Potentials
for the use of pyridoxyl derivatives in the synthesis of linear, multiplet, and cyclic peptides are discussed. 相似文献
7.
S. L. Grokhovsky 《Molecular Biology》2006,40(2):276-283
Cleavage of double-stranded DNA fragments with known nucleotide sequences upon sonication at 22 and 44 kHz was studied by PAGE. The cleavage rate was shown to depend on the fragment size, pH, ionic strength, and temperature. Double-strand breaks occurred preferentially in 5′-CpG-3′ dinucleotides. The strand was broken between C and G so that the phosphate group was at the 5′ side of G in the products. The cleavage rate proved to depend on the sequences flanking the cleavage site. The character of cleavage changed in the presence of Pt-bis-netropsin, a sequence-specific ligand that alters the local conformation of DNA. 相似文献
8.
Regulation of the synthesis of human chorionic gonadotropin by strains of HeLa cells in culture 总被引:2,自引:0,他引:2
Janice Yang Chou 《In vitro cellular & developmental biology. Plant》1978,14(9):775-778
Summary Thirty-seven strains of HeLa cells were examined for their ability to synthesize human chorionic gonadotropin (hCG) and its
alpha subunit (hCG-α) in culture. Synthesis of hCG-α and hCG also was investigated in the presence of sodium butyrate and
5-bromo-2′-deoxyuridine (BrdUrd). All HeLa strains synthesized hCG-α in culture. Sodium butyrate increased the synthesis of
hCG-α in all HeLa cells; BrdUrd increased synthesis in 32 of the 37 strains examined. Although few HeLa strains synthesized
hCG in the absence of inducers, hCG was detected in most strains in the presence of sodium butyrate. The synthesis of hCG
and its alpha subunit is, therefore, a stable genetic characteristics of HeLa cells.
Certain preparations of hCG and its subunits were generously provided through the Center for Population Research of the National
Institute of Child Health and Human Development, NIH. 相似文献
9.
F. M. Pisani Mariarita De Felice Giuseppe Manco Mosè Rossi 《Extremophiles : life under extreme conditions》1998,2(3):171-177
DNA polymerase from Sulfolobus solfataricus, strain MT4 (Sso DNA pol), was one of the first archaeal DNA polymerases to be isolated and characterized. Its encoding gene
was cloned and sequenced, indicating that Sso DNA pol belongs to family B of DNA polymerases. By limited proteolysis experiments
carried out on the recombinant homogeneous protein, we were able to demonstrate that the enzyme has a modular organization
of its associated catalytic functions (DNA polymerase and 3′-5′ exonuclease). Indeed, the synthetic function was ascribed
to the enzyme C-terminal portion, whereas the N-terminal half was found to be responsible for the exonucleolytic activity.
In addition, partial proteolysis studies were utilized to map conformational changes on DNA binding by comparing the cleavage
map in the absence or presence of nucleic acid ligands. This analysis allowed us to identify two segments of the Sso DNA pol
amino acid chain affected by structural modifications following nucleic acid binding: region 1 and region 2, in the middle
and at the C-terminal end of the protein chain, respectively. Site-directed mutagenesis studies will be performed to better
investigate the role of these two protein segments in DNA substrate interaction.
Received: January 22, 1998 / Accepted: February 16, 1998 相似文献
10.
Synthesis of cellulose in vitro is expected to afford tailor-made cellulosic materials with highly homogeneous structure compared to natural cellulosic materials. Here we report the enzymatic synthesis of cellulose II with high crystallinity from glucose and α-glucose 1-phosphate (αG1P) by cellodextrin phosphorylase (CDP). Although glucose had been believed not to act as a glucosyl acceptor of CDP, a significant amount of insoluble cellulose was precipitated without accumulation of soluble cello oligosaccharides when glucose was mixed with αG1P and CDP. This phenomenon can be explained in terms of the large difference in acceptor reactivity between glucose and cello oligosaccharides. 1H NMR spectrometric analysis revealed that this insoluble cellulose had an average degree of polymerization (DP) of nine. TEM observation, together with electron and X-ray diffraction studies, indicated that the insoluble cellulose formed platelet-shaped single lamellar crystals of cellulose II, several μm in length and several hundred nm in width; this is large compared to reported cellulose crystals. The thickness of the lamellar crystal is 4.5 nm, which is equivalent to a chain length of a cello oligosaccharide with DP nine and is consistent with the 1H NMR spectroscopic results. These results suggest that cello oligosaccharides having an average DP of nine are synthesized in vitro by CDP when glucose is used as an acceptor, and the product forms highly crystalline cellulose II when it precipitates. 相似文献
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QBEND/10 is a mouse immunoglobulin lambda-chain monoclonal antibody with strict specificity against human hematopoietic progenitor cell antigen CD34. Our in vitro study showed that QBEND/10 impairs the tube formation of human umbilical vein endothelial cells (HUVECs), suggesting that the antibody may be of potential benefit in blocking tumor angiogenesis. We provided a de novo protein sequencing method through tandem mass spectrometry to identify the amino acid sequences in the variable heavy and light chains of QBEND/10. To reduce immunogenicity for clinical applications, QBEND/10 was further humanized using the resurfacing approach. We demonstrate that the de novo sequenced and humanized QBEND/10 retains the biological functions of the parental mouse counterpart, including the binding kinetics to CD34 and blockage of the tube formation of the HUVECs. 相似文献
14.
L. E. M. Nery M. A. da Silva A. M. Lauro Castrucci 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1998,168(8):624-630
The participation of cyclic nucleotide-dependent intracellular signalling pathways in the pigment translocation induced by
pigment-dispersing hormone (α -PDH) or pigment-concentrating hormone (PCH) was investigated in the erythrophores of the freshwater
shrimp, Macrobrachium potiuna. Cholera toxin, forskolin and dibutyryl cyclic adenosine 3′5′ monophosphate (dbcAMP) were able to induce pigment dispersion
with effective agonist concentrations for half maximal response (EC50 s) of 2.8 · 10−11 mol · l−1, 7.0 · 10−7 mol · l−1 and 3.3 · 10−7 mol · l−1, respectively. KT5720 (10−7 mol · l−1 and 10−6 mol · l−1) significantly shifted the dose response curve to α -PDH to the right. Dibutyryl cyclic guanosine 3′5′ monophosphate (dbcGMP)
was ineffective in inducing either pigment aggregation or dispersion. 2′5′ dideoxyadenosine (DDA) and SQ22,536 essentially
elicit a pigment-aggregating response in a dose-dependent manner. These effects were not due to the activation of purinergic
receptors, since concentrations up to 10−4 mol · l−1 of adenosine and adenosine triphosphate (ATP), and up to 10−3 mol · l−1 of uracil triphosphate (UTP) did not elicit pigment aggregation. In order to verify if PCH decreased cyclic adenosine 3′5′
monophosphate (cAMP) levels, cumulative dose-response curves to PCH in the absence and presence of pertussis toxin and 8-MOM-IBMX
were determined. However, neither drug significantly affected PCH activity. The levels of cAMP in the integument cells of
M. potiuna were significantly increased (P < 0.05) by α -PDH (10−7 mol · l−1) and forskolin (10−6 mol · l−1), but were not affected by PCH (10−7 or 10−10 mol · l−1). In conclusion, α -PDH seems to elicit pigment dispersion through the activation of a Gs-protein coupled receptor resulting
in cAMP increase and cAMP-dependent protein kinase (PKA) activation. Furthermore, although a decrease in cAMP was assumed
to be responsible in turn for the action of PCH, such a decrease could not be directly demonstrated.
Accepted: 11 August 1998 相似文献
15.
Effective methods of the directed introduction of diphosphoryl disulfide bridges into hairpin DNA duplexes in place of natural
phosphodiester groups were developed using the H2O2-effected ligation of 3′- and 5′-thiophosphorylated oligonucleotides or by autoligation of a preactivated oligonucleotide
derivative with a phosphorothioate-bearing oligomer. The postsynthetic recombination of the disulfide-linked oligonucleotide
fragments was characterized. It was shown that, along with template-directed reactions, out-of-duplex formation and exchange
of diphosphoryl disulfide bonds in the DNA sugar-phosphate backbone may occur. In modified hairpin DNA, a spontaneous exchange
of disulfide-linked fragments virtually does not take place because of the intramolecular duplex formation. 相似文献
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Adolph J. Ferro Arther A. Vandenbark Kevin Marchitto 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,588(3):294-301
To determine if increased 5′-methylthioadenosine phosphorylase activity in activated lymphocytes may be responsible for the decreased inhibitory effect noted when 5′-methylthioadenosine is added after stimulation, the activity of this enzyme was monitored during lymphocyte transformation. A direct correlation existed between the transformation process and 5′-methylthioadenosine phosphorylase activity; the longer the stimulation process progressed, the greater the enzyme activity. The 7-deaza analog of 5′-methylthioadenosine, 5′-methylthiotubercidin, was utilized to explore further the role that the phosphorylase may play in the reversal process. 5′-Methylthioadenosine acted as a potent inhibitor, but not a substrate, of the 5′-methylthioadenosine phosphorylase, and was an even more potent inhibitor of lymphocyte transformation than 5′-methylthioadenosine. However, in direct contrast to the 5′-methylthioadenosine effect, inhibition by 5′-methylthiotubercidin could not be completely reversed. These data suggest the 5′-methylthioadenosine phosphorylase plays an important role in reversing 5′-methylthioadenosine-mediated inhibition and that the potent, nonreversible inhibitory effects of 5′-methylthiotubercidin are due to its resistance to 5′-methylthioadenosine phosphorylase degradation. 相似文献
18.
De novo purine biosynthesis in the crustacean Artemia: Influence of salinity and geographical origin
Antonio Liras Pedro Rotllán Pilar Llorente 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1992,162(3):263-266
Summary In vivo studies of the incoporation of [U-14C]glycine into purine nucleotides have established the de novo pathway for purine biosynthesis in Artemia sp. during the early period of larval development. This pathway can be modified by the salt concentration of the incubation media. In addition, Artemia of different geographical origins may differ with respect to the detection, functionality and variability of this metabolical pathway.Abbreviations ADP
adenosine, diphosphate
- ASN
acid soluble nucleotides
- ATP
adenosine triphosphate
- DNA
desoxyribonucleic acid
- GDP
guanosine diphosphate
- GP4G
pl, p4-diguanosine 5-tetraphosphate
- HPLC
high performance liquid chromatography
- PCA
perchloric acid
- RNA
ribonucleic acid 相似文献
19.
Stasolla C Loukanina N Ashihara H Yeung EC Thorpe TA 《Journal of plant physiology》2007,164(4):429-441
Changes in the pattern of pyrimidine nucleotide metabolism were investigated in Pinus radiata cotyledons cultured under shoot-forming (SF; +N(6)-benzyladenine) and non-shoot-forming (NSF, -N(6)-benzyladenine) conditions, as well as in cotyledons unresponsive (OLD) to N(6)-benzyladenine. This was carried out by following the metabolic fate of externally supplied (14)C-labeled orotic acid, intermediate of the de novo pathway, and (14)C-labeled uridine and uracil, substrates of the salvage pathway. Nucleic acid synthesis was also investigated by following the metabolic fate of (14)C-labeled thymidine during shoot bud formation and development. The de novo synthesis of pyrimidine nucleotides was operative under both SF and NSF conditions, and the activity of orotate phosphoribosyltransferase (OPRT), a key enzyme of the de novo pathway, was higher in SF tissue. Utilization of both uridine and uracil for nucleotide and nucleic acid synthesis clearly indicated that the salvage pathway of pyrimidine metabolism is also operative during shoot organogenesis. In general, uridine was a better substrate for the synthesis of salvage products than uracil, possibly due to the higher activity of uridine kinase (UK), compared to uracil phosphoribosyltransferase (UPRT). Incorporation of uridine into the nucleic acid fraction of OLD cotyledons was lower than that observed for their responsive (day 0) counterparts. Similarly, uracil utilization for nucleic acid synthesis was lower in NSF cotyledons, compared to that observed for SF tissue after 10 days in culture. This difference was ascribed to higher UPRT activity measured in the latter. Thus, there was an apparent difference in the utilization of nucleotides derived from uracil and uridine for nucleotide synthesis. The increased ability to produce pyrimidine nucleotides via the salvage pathway during shoot bud formation may be required in support of nucleic acid synthesis occurring during the process. Studies on thymidine metabolism confirmed this notion. 相似文献
20.
Edible mushroom fungi in the genera Lyophyllum, Tricholoma, Leucopaxillus, Suillus, Rhizopogon, Lactarius, and Morchella were tested for mycorrhization with Pinus densiflora in vitro. Most of the tested fungi in the genera Lyophyllum, Tricholoma, Suillus, Rhizopogon, and Lactarius formed ectomycorrhizas 2–4 months after fungal inoculation. Mycorrhizal seedlings were then acclimatized in open-pot soil
under growth-chamber conditions. Almost all mycorrhizal seedlings sustained their symbiont and developed new mycorrhizas for
8–9 months after transplantation. Under these conditions, more than half of the tested species formed primordia and Tricholoma flavovirens, Rhizopogon rubescens, and Lactarius akahatsu developed basidiocarps with young host plants.
Accepted: 28 November 2000 相似文献