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1.
《Insect Biochemistry》1976,6(3):333-337
Thymidylate synthetase activity (as expressed per mg of protein and per the whole insect) in extracts from Bombyx mori changes considerably during development. The enzyme is present in unfertilized eggs. After fertilization its activity initially increases but then decreases in parallel with the beginning of diapause. During postdiapause embryonic development the level of the enzyme quickly increases, but decreases again after hatching. In larvae the activity is low during the first instars but rises dramatically after the 4th ecdysis. The whole period of pharate pupal, pupal, and pharate adult development is characterized by a U-shaped pattern of the thymidylate synthetase activity.In order to explain some aspects of this pattern the activity of the thymidylate synthetase in extracts from some organs of the silkworm was also examined. 相似文献
2.
《The International journal of biochemistry》1993,25(5):671-680
- 1.1. Changes in the spectrum of pyridoxal phosphate (PLP) were produced by adding an equimolar amount of native thymidylate synthase, but not by adding denatured enzyme or enzyme modified by sulfhydryl-blocking reagents.
- 2.2. The dissociation constant of the thymidylate synthase-PLP complex determined by equilibrium dialysis was 9 ± 1.6 μM, the maximum number of PLP molecules bound per molecule of native thymidylate synthase was 2.5 ± 0.4, and the Hill coefficient was 0.97.
- 3.3. No evidence of PLP binding was found with denatured thymidylate synthase, and only slight binding was observed when enzyme SH groups were blocked or when the active site was blocked with 5-fluorodeoxyuridylate (FdUMP) and methylenetetrahydrofoliate.
- 4.4. The presence of dUMP, dTMP, or FdUMP interfered with the binding of PLP to thymidylate synthase, and the presence of equimolar amounts of PLP interfered with the binding of dUMP.
3.
Thymidylate synthase activity from chlamydomonas cells and cultured tissues of Nicotiana, pinus, and daucus
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Prior preparation of N(5),N(10)-methylenetetrahydrofolate from dl-tetrahydrofolate and [(14)C]formaldehyde resulted in an improved assay for thymidylate synthase. Although preparations from tobacco seedlings and cotton root tips (0.25 centimeter) were inconsistent with respect to enzyme activity, extracts from actively growing cell cultures of Chlamydomonas, Nicotiana hybrid callus, Pinus callus, and Daucus proembryonic cells contained significant levels of thymidylate synthase (12.3 to 23.8 nanomoles of thymidylate formed per milligram of protein per hour). 相似文献
4.
D Rosson H B Otwell R B Dunlap 《Biochemical and biophysical research communications》1980,97(2):500-505
Sulfhydryl-blocked thymidylate synthetase (EC 2.1.1.4.5) is rapidly inactivated by low concentrations of tetranitromethane. This reagent first nitrates two non-essential tyrosines per dimeric enzyme molecule followeed by two essential tyrosines with no oxidation of sulfhydryl groups. dUMP affords significant protection against inactivation. These results suggest that essential tyrosyl residues are present in the active sites of the enzyme. 相似文献
5.
M Gálová D Koptidesová D Rusznáková P Racay M Kollárová 《Archives of biochemistry and biophysics》1992,296(1):81-87
NADPH-dependent thymidylate synthetase from Streptomyces aureofaciens has been purified to homogenity by a two-step chromatographic procedure including anion-exchange chromatography and affinity chromatography on methotrexate-Sepharose 4B. The enzyme was purified 1025-fold with a 34% yield. Basic characteristics of the enzyme were determined: molecular weight of the enzyme subunit (28,000), pH and temperature optimum, effect of cations, dependency on reducing agents, Km values for dUMP, mTHF, and NADPH (3.78, 21.1, and 38.9 microM, respectively), and inhibition effect of 5-FdUMP. Binding studies revealed the enzyme mechanism to be ordered sequential: dUMP bound before mTHF. S. aureofaciens thymidylate synthetase exhibits an absolute requirement for NADPH for the enzyme activity--a unique feature not displayed by any of the thymidylate synthetases isolated so far. NADPH is not consumed during enzyme reaction, indicating its regulatory role. The properties of S. aureofaciens thymidylate synthetase show that it is a monofunctional bacterial enzyme. 相似文献
6.
Nancy J. Carpenter 《Genetics》1973,75(1):113-122
Thymidylate synthetase has been examined with respect to the normal pattern of activity throughout the development of the Oregon-R strain of Drosophila melanogaster. Large amounts of the enzyme are present in both the unfertilized and the fertilized eggs. A comparison of the ovarian thymidylate synthetase activity of the Oregon-R strain and the female sterility mutants, fs(2)B, fu, and fs(1)N, indicates variations in the activity of this enzyme in each strain. At four days of age, the ovarian-specific activity of the female sterility mutants is comparable to or less than that of the Oregon-R strain, but it is reduced at fifteen days of age. The enzyme activity per ovary is low in the fs(2)B strain but is similar in the Oregon-R, fu, and fs(1)N strains. When expressed as activity per organism, thymidylate synthetase declines after six to eight hours of development until the minimal level is reached in the late embryonic stage. Enzyme activity rises throughout the larval instars, reaching a maximum immediately after puparium formation. The activity decreases during pupation, but rises again during the first four days of adult life. 相似文献
7.
Association of thymidylate kinase activity with pyrimidine deoxyribonucleoside kinase induced by herpes simplex virus. 总被引:11,自引:0,他引:11
Thymidine kinase derived from LMTK+ does not exhibit thymidylate kinase activity. However, protein isolated by affinity column chromatography from thymidine kinase-deficient mouse cells (LMTK-) infected by herpes simplex virus type 1 shows thymidylate kinase activity in addition to thymidine kinase and deoxycytidine kinase activities. The virus-induced multifunctional enzyme has a molecular weight of 85,000, whereas the molecular weight of thymidylate kinase from uninfected LMTK- mouse cells is 71,000. The virus-induced enzyme has a Km for thymidine of 0.8 micromolar, and for thymidylate of 25 micromolar, and for thymidylate of 25 micromolar; the ratio of Vmax for thymidylate kinase to thymidine kinase is 1.7. When subjected to isoelectric focusing, thymidylate kinase activity is not separated from thymidine kinase activity, and even though four peaks of activity are observed they have a constant ratio of thymidylate kinase to thymidine kinase activity. The isoelectric points (pI) of these four peaks are 4.8, 5.8, 6.2, and 6.6, respectively. Thymidylate kinase, derived from uninfected cells when subjected to isoelectric focusing, separates into a major component with an isoelectric point at pH 8.2 and a minor component at pH 7.7. Although thymidine and thymidylate kinase activities derived from the virus-infected cells cannot be separated either by affinity column chromatography, glycerol density gradient centrifugation, or isoelectric focusing, there is a differential rate of inactivation when the enzyme is subjected to incubation at 37 degrees, with thymidylate kinase activity being more labile than thymidine kinase activity. 相似文献
8.
Relationship Between Escherichia coli B Titer and the Level of Deoxycytidylate Deaminase Activity Induced on Bacteriophage T2r+ Infection
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The activities of six bacteriophage T2r(+)-induced enzymes (thymidylate synthetase, deoxycytidylate deaminase, thymidylate kinase, deoxycytidylate hydroxymethylase, deoxycytidine pyrophosphatase, and dihydrofolate reductase) were measured after dilution of phage-infected Escherichia coli B from 8 x 10(8) to 2 x 10(8) cells per ml. The only enzyme activity altered was that of deoxycytidylate deaminase, which increased three- to fourfold. Conversely, the rapid concentration of cells from 2 x 10(8) to 8 x 10(8) per ml did not result in a reduction in deaminase activity. Although an enhancement in aeration reduced the response of deoxycytidylate deaminase to cellular dilution, the influence of potential metabolic inhibitors or activators could not be shown. The change in deoxycytidylate deaminase activity appeared to be associated with an altered translational event, since the increase could not be prevented by rifampin but was blocked effectively by chloramphenicol and hydroxylamine. In addition, antibody to the T2 phage-induced deoxycytidylate deaminase demonstrated that the increase in enzyme activity was associated with a corresponding increase in radioactive leucine incorporated into the enzyme antigen. 相似文献
9.
Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs. 相似文献
10.
Hideaki Yamada Osao Adachi Koichi Ogata 《Bioscience, biotechnology, and biochemistry》2013,77(10):912-917
There was an increase in copper content proportional to the increase in specific activity of the enzyme during the purification of amine oxidase of Aspergillus niger. The recrystallized enzyme preparation contained 1 g atom of copper per 83,000 g of protein. This indicated that the enzyme contained 3 g atoms of copper per mole of enzyme. The copper in the enzyme was removed by dialysis against sodium diethyldithiocarbamate with concomitant loss of activity. The dialyzed enzyme was reactivated by the addition of cupric copper.The copper in the enzyme was present in cupric state. No valency change of the copper was observed in the catalytic activity.The enzyme was inhibited by various chelating agents, and potently inhibited by various carbonyl reagents. 相似文献
11.
Deoxycytidine Triphosphate Deaminase: Characterization of an Escherichia coli Mutant Deficient in the Enzyme 总被引:16,自引:13,他引:3
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G. A. O''Donovan G. Edlin J. A. Fuchs J. Neuhard E. Thomassen 《Journal of bacteriology》1971,105(2):666-672
A mutant of Escherichia coli, previously shown to contain abnormal nucleoside triphosphate pools, was found to be defective in its ability to synthesize thymidine nucleotides. The defect is not in the enzyme thymidylate synthetase but in deoxycytidine triphosphate deaminase, an enzyme that supplies deoxyuridine monophosphate, the substrate for thymidylate synthetase. 相似文献
12.
Human thymidylate synthase [EC 2.1.1.45] was purified to homogeneity and its NH2-terminal amino acid sequence was determined taking advantage of the following facts: i) The source of the enzyme was a transformant of mouse FM3A mutant cells which lacks mouse thymidylate synthase but overproduces human thymidylate synthase. ii) The enzyme could be purified on two kinds of affinity column, Cibacron blue dye-bound agarose and methotrexate-bound Sepharose. iii) The enzyme could finally be separated from a trace of impurities by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate. The purified human thymidylate synthase had a subunit with a molecular weight of 33,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was subjected to Edman degradation and the NH2-terminal 24 amino acids were sequenced by successive use of a high-sensitivity gas-phase protein sequencer and high performance liquid chromatography to be as follows: Pro-Val-Ala-Gly-Ser-Glu-Leu-Pro-Arg-Arg-Pro-Leu-Pro-Pro-Ala-Ala-Gln-Glu- Arg-Asp -Ala-Glu-Pro-Arg-. 相似文献
13.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(2):149-156
AbstractThree vitamin B6 analogues have been synthesized and tested as inhibitors of thymidylate synthase. The compounds are: 4′,5′-dichloro-, 4,5′-dibromo- and 4′, 5′-diiodo-pyridoxine. All three analogues inhibited the enzyme irreversibly. The kinetic data for the chloro- and bromo-analogues showed that a limiting rate of inhibition is approached as the inhibitor concentration is increased, which indicates that a reversible enzyme: inhibitor affinity complex is formed prior to the irreversible reaction. 4′,5′-Dibromo-pyridoxine exhibited a greater binding affinity (lower Ki) for thymidylate synthase than 4′,5′-dichloro-pyridoxine, and it also reacted faster to irreversibly inhibit the enzyme. The presence of the substrate dUMP (10μM) completely protected thymidylate synthase from inhibition. These data suggest that the halogenated vitamin B6 analogues are active site-directed inhitors of thymidylate synthase, which first bind reversibly to the catalytic site and then react irreversibly with the enzyme. 相似文献
14.
H Ahmed J L Aull D E Williams S D Worley 《The International journal of biochemistry》1986,18(3):245-250
The effects of two chlorine disinfectants, calcium hypochlorite (HTH) and 3-chloro-4,4-dimethyl-2-oxazolidinone (Agent I), on the activity of thymidylate synthase have been investigated. Although both disinfectants inactivated the enzyme, the following differences were observed: When the two disinfectants were used at the same total chlorine concentration, the rate and extent of inactivation were greater with Agent I than HTH. The substrate dUMP partially protected thymidylate synthase from inactivation by Agent I, but it did not appreciably protect against inactivation by HTH. Large changes in the ultraviolet spectrum of the enzyme occurred when it was treated with HTH, which suggests reactions with aromatic amino acid side chains; no spectral changes occurred when thymidylate synthase was treated with Agent I. Blocking the sulfhydryl groups of thymidylate synthase with sulfhydryl reagents prevented the irreversible inactivation of the enzyme by Agent I, but not by HTH. 相似文献
15.
Site-directed mutagenesis of arginine 179 of thymidylate synthase. A nonessential substrate-binding residue 总被引:2,自引:0,他引:2
D V Santi K Pinter J Kealey V J Davisson 《The Journal of biological chemistry》1990,265(12):6770-6775
X-ray structural studies have shown that Arg-179 of thymidylate synthase is complexed to bound inorganic phosphate or to the 5'-phosphate of the bound substrate dUMP. The importance of Arg-179 to the structure/function of thymidylate synthase is also indicated by its complete conservation among the 17 thymidylate synthases thus far sequenced. In the present work, Arg-179 has been replaced by Thr, Ala, Lys, and Glu using site-directed mutagenesis with a mixture of four synthetic oligonucleotides as primers. The mutant proteins complement thymidylate synthase-deficient Escherichia coli and show high enzyme activity. Each of these mutants has been purified to homogeneity, partially sequenced to verify the mutation, and has had its steady state kinetic parameters determined. The most significant effect of all mutations is localized to a decrease in the net rate of association of thymidylate synthase with dUMP; the Lys mutant also shows an apparent increase in the dissociation constant of the folate cofactor of the reaction. The high activity in the mutant enzymes is explained by "plasticity" of the enzyme and compensatory actions of the other Arg residues. Why the Arg-179 residue has been conserved during evolution remains an open question. 相似文献
16.
Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule. 相似文献
17.
Cieśla J Gołos B Wałajtys-Rode E Jagielska E Płucienniczak A Rode W 《Acta biochimica Polonica》2002,49(3):651-658
Mouse thymidylate synthase R209K (a mutation corresponding to R218K in Lactobacillus casei), overexpressed in thymidylate synthase-deficient Escherichia coli strain, was poorly soluble and with only feeble enzyme activity. The mutated protein, incubated with FdUMP and N(5,10)-methylenetetrahydrofolate, did not form a complex stable under conditions of SDS/polyacrylamide gel electrophoresis. The reaction catalyzed by the R209K enzyme (studied in a crude extract), compared to that catalyzed by purified wild-type recombinant mouse thymidylate synthase, showed the K(m) value for dUMP 571-fold higher and V(max) value over 50-fold (assuming that the mutated enzyme constituted 20% of total crude extract protein) lower. Thus the ratios k(cat, R209K)/k(cat, 'wild') and (k(cat, R209K)/K(m, R209K)(dUMP))/( k(cat, 'wild')/K(m, 'wild')(dUMP)) were 0.019 and 0.000032, respectively, documenting that mouse thymidylate synthase R209, similar to the corresponding L. casei R218, is essential for both dUMP binding and enzyme reaction. 相似文献
18.
B P Sani A Vaid J G Cory R W Brockman R D Elliott J A Montgomery 《Biochimica et biophysica acta》1986,881(2):175-184
5'-Bromoacetamido-5'-deoxythymidine (BAT), 5'-iodoacetamido-5'-deoxythymidine (IAT), 5'-chloroacetamido-5'-deoxythymidine (CAT) and [14C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were investigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT greater than IAT greater than CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4-8-fold higher than it was in the absence of dUMP. The I50 values for BAT were 1 X 10(-5) M and 1.2 X 10(-6) M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thymidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The Km for the enzyme was 9.2 microM, and the Ki determined for competitive inhibition by BAT was 5.4 microM. Formation of a tight, irreversible complex is inferred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min-1, and the steady-state constant of inactivation, Ki, was estimated to be 6.6 microM. The 5'-haloacetamido-5'-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism. 相似文献
19.
Regulation of thymidylate synthase gene expression in mouse fibroblasts synchronized by mitotic selection 总被引:2,自引:0,他引:2
Previous studies have shown that thymidylate synthase gene expression is regulated over a wide range in response to growth stimulation in cultured mouse fibroblasts. In the present study we show that the gene is also regulated during the cell cycle in continuously growing cells. Our analyses were conducted with a fluorodeoxyuridine-resistant mouse 3T6 cell line that overproduces thymidylate synthase and its mRNA by a factor of 50 due to gene amplification. Cells were synchronized by mitotic selection. RNA blot analyses showed that the amount of thymidylate synthase mRNA increased 5- to 10-fold as cells progressed from G1 through the middle of S phase. S1 nuclease protection assays showed that the pattern of 5' termini of thymidylate synthase mRNA was the same in G1 and S phase. Despite the large increase in thymidylate synthase mRNA content, the level of the enzyme increased only by a factor of 2 as cells progressed from G1 to mid S phase. This apparent discrepancy can be explained by the fact that the enzyme is highly stable. 相似文献
20.
Human leukemic cells resistant to 5-fluoro-2'-deoxyuridine contain a thymidylate synthetase with lower affinity for nucleotides 总被引:4,自引:0,他引:4
A line of human lymphocytic leukemia cells (CCRF-CEM) has been obtained which is 140-fold resistant to the potent cell growth inhibitor 5-fluoro-2'-deoxyuridine (FdUrd). The cells were also 11-fold cross-resistant to 5-fluorouracil. In contrast to several previous studies involving FdUrd-resistant mouse cells, thymidylate synthetase levels were not substantially elevated in these FdUrd-resistant human leukemic cells. Thymidine kinase activity was also unchanged in the resistant cells, although the levels of 5-fluoro-2'-deoxyuridylate (FdUMP), the potent inhibitor of thymidylate synthetase, generated at equimolar doses of FdUrd were about 40% lower than in the sensitive cells. Studies of the kinetics of FdUMP binding to thymidylate synthetase isolated from the FdUrd-resistant cells disclosed a considerably higher dissociation constant (Kd = 1.0 X 10(-9) M) for the ternary covalent enzyme . FdUMP . 5,10-methylene tetrahydrofolate complex compared to the value obtained with enzyme from sensitive cells (Kd = 4.4 X 10(-11) M). The thymidylate synthetase from the FdUrd-resistant cells also showed 17-fold weaker binding of 2'-deoxyuridylate, even though the Km value for 2'-deoxyuridylate was 3-fold lower compared to the enzyme from FdUrd-sensitive cells. The turnover number of the altered enzyme was 1.8-fold higher than that for the normal enzyme but the rate constants for the release of FdUMP from the ternary complex, which is also an enzyme-catalyzed reaction, were identical for both enzymes. Electrophoresis of the radiolabeled ternary complexes on nondenaturing gels showed small but reproducible differences in migration rates. These results demonstrate that the mechanism of resistance to FdUrd in this cell line involves an alteration in the target enzyme, thymidylate synthetase, which causes it have a lower affinity for nucleotides. 相似文献