scholarly journals Regulation of Thymidylate Synthetase Activity in Cultured Mammalian Cells

1972 ◽  
Vol 10 (2) ◽  
pp. 471-486 ◽  
Author(s):  
ABIGAIL H. CONRAD ◽  
F. H. RUDDLE
Keyword(s):  
Enzyme Activity ◽  
Cell Division ◽  
Mammalian Cells ◽  
Actinomycin D ◽  
Specific Activity ◽  
Thymidylate Synthetase ◽  
Lag Phase ◽  
Synthetase Activity ◽  
Pronounced Increase ◽  
Culture Cycle

Changes in thymidylate synthetase specific activity in Don Chinese hamster cells grown in vitro have been examined during the culture cycle and after exposure of lag- and log-phase cultures to drugs which inhibit DNA, RNA, and protein synthesis. During the culture cycle enzyme activity was low during lag phase, rose 6- to 8-fold before log phase, fluctuated between 5.5 and 9 nmol dTMP/h/107 cells during log phase, and declined to base level during stationary phase. Puromycin prevented all increases in enzyme specific activity and caused a decrease in enzyme activity when applied to log-phase cultures. Actinomycin D prevented the initial rise in enzyme activity if applied during early lag phase but caused a pronounced increase in enzyme activity above control levels when applied during log phase. High thymidine concentration (1 mM) stopped cell division in log-phase cultures but did not alter the log-phase plateau level of thymidylate synthetase activity. Fluorodeoxyuridine stopped cell division and depressed enzyme activity to varying degrees depending upon its concentration, but at concentrations less than 10-6M enzyme activity eventually returned to normal log-phase levels and cell division resumed if puromycin was not present. Methotrexate stopped cell division and caused a 3- to 4-fold increase in enzyme activity above control levels if puromycin was not present. This increase occurred in the presence of actinomycin D but was retarded by addition of thymidine when actinomycin D was not present. These experiments suggest that the regulation of thymidylate synthetase activity in log-phase cells is complex and may involve thymidine triphosphate.

scholarly journals THYMIDYLATE SYNTHETASE IN MUTANTS OF DROSOPHILA MELANOGASTER

Genetics ◽  
1973 ◽  
Vol 75 (1) ◽  
pp. 113-122
Author(s):  
Nancy J Carpenter
Keyword(s):  
Enzyme Activity ◽  
Drosophila Melanogaster ◽  
Specific Activity ◽  
Adult Life ◽  
Thymidylate Synthetase ◽  
Female Sterility ◽  
Synthetase Activity ◽  
Normal Pattern ◽  
Puparium Formation ◽  
Late Embryonic Stage

ABSTRACT 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.

scholarly journals Thymidylate synthetase activity in bone marrow cells in pernicious anemia

Blood ◽  
1975 ◽  
Vol 46 (5) ◽  
pp. 699-704
Author(s):  
S Sakamoto ◽  
M Niina ◽  
F Takaku
Keyword(s):  
Bone Marrow ◽  
Enzyme Activity ◽  
Pernicious Anemia ◽  
Bone Marrow Cells ◽  
Normal Subjects ◽  
Thymidylate Synthetase ◽  
Synthetase Activity ◽  
Release Assay ◽  
Methylene Tetrahydrofolate ◽  
Marrow Cells

The tritium release assay for the demonstration of thymidylate synthetase activity has been applied to the measurement of enzyme activity in the bone marrow of four patients with pernicious anemia and nine normal subjects. On the average, an approximately ninefold increase in enzyme activity was observed in patients with pernicious anemia. In the absence of 5, 10-methylene-tetrahydrofolate, enzyme activity was reduced in both normal and in pernicious anemia cells. Addition of 5, 10-methylene-tetrahydrofolate to the assay medium resulted in a far greater activation of thymidylate synthetase activity in megaloblastic bone marrow cells than in the cells of control subjects.

Thymidine-requiring mutants of Dictyostelium discoideum

1984 ◽  
Vol 4 (12) ◽  
pp. 2784-2791
Author(s):  
G Podgorski ◽  
R A Deering
Keyword(s):  
Dictyostelium Discoideum ◽  
Nuclear Dna ◽  
Specific Activity ◽  
High Specific Activity ◽  
Normal Growth ◽  
Thymidylate Synthetase ◽  
Synthetase Activity ◽  
Cell Extracts ◽  
Dna Replication And Repair

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.

Purification and Properties of Thymidylate Synthetase from Pig Thymus

1972 ◽  
Vol 50 (4) ◽  
pp. 352-362 ◽  
Author(s):  
V. S. Gupta ◽  
J. B. Meldrum
Keyword(s):  
Catalytic Activity ◽  
Sulfonic Acid ◽  
Specific Activity ◽  
Gel Filtration ◽  
Thymidylate Synthetase ◽  
Heat Inactivation ◽  
Synthetase Activity ◽  
Purification And Properties ◽  
Michaelis Constants ◽  
Sulfhydryl Compounds

Thymidylate synthetase of pig thymus has been separated into two principal forms (designated I and II, based on their order of elution) by chromatography on CM-Sephadex. By the use of (NH4)2SO4 the synthetase activity was separated into two fractions, and these were further purified by gel filtration using Sephadex G-100 and chromatography on CM-Sephadex. The highest specific activity obtained for I and II was 10.4 and 16.3 μmol of thymidine-5′-phosphate per hour per milligram of protein at 25° and pH 7.3 which represents a purification of 1680- and 2630-fold, respectively. Electrophoretically, I and II appear to be 70–80% pure. The Michaelis constants of 7.4 × 10−6 M, 1.7 × 10−5 M, and 1.8 × 10−4 M for II with respect to deoxyuridine-5′-phosphate, 5,10-methlenetetrahydrofolate, and uridine-5′-phosphate, respectively, have been determined. A double pH optima in the range of 6.6–6.8 and 7.2–7.4 in 2-N-morpholinoethane sulfonic acid buffer was exhibited by both forms. Forms I and II showed maximal catalytic activity only in the presence of sulfhydryl compounds (60 mM) and also had the ability to methylate uridine-5′-phosphate, although at a slower rate (ca. 28% and 13%, respectively) compared with the rate of methylation of deoxyuridine-5′-phosphate. Both deoxyuridine-5′-phosphate and tetrahydrofolate (to a lesser extent) afforded protection to II against heat inactivation.

Glutamine synthetase from Mycobacterium avium

1984 ◽  
Vol 30 (3) ◽  
pp. 353-359 ◽  
Author(s):  
Maria E. Alvarez ◽  
C. M. McCarthy
Keyword(s):  
Enzyme Activity ◽  
Glutamine Synthetase ◽  
Nitrogen Source ◽  
Mycobacterium Avium ◽  
Specific Activity ◽  
Sedimentation Coefficient ◽  
Ammonia Assimilation ◽  
Methionine Sulfoximine ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity

Mycobacterium avium was previously shown to be dependent upon ammonia or glutamine as a nitrogen source. In an effort to assess the physiology of ammonia assimilation by M. avium, a characterization of its glutamine synthetase was performed. The enzyme from M. avium was purified by streptomycin sulfate treatment, ammonium sulfate precipitation, and affinity chromatography. The enzyme was unusual in that it had a pH optimum of 6.4 and maximum enzyme activity was obtained between 50 and 60 °C as shown by the transferase assay. The glutamine synthetase activity from batch-cultured cells decreased with increasing concentration of ammonium chloride in the range of 0.25–5 μ mol/mL of medium, which demonstrated a response to environmental supply of a nitrogen source. The mycobacterial enzyme was similar to the other bacterial glutamine synthetases in terms of molecular weight and sedimentation coefficient which were 600 000 and 19.5 S, respectively, and enzyme activity was lost by treatment with a glutamate analog, methionine sulfoximine. The isoelectric point was, however, pH 4.5. Treatment of the enzyme with snake venom phosphodiesterase resulted in an increase in specific activity. AMP was released by the phosphodiesterase treatment, thus demonstrating that M. avium glutamine synthetase was regulated by adenylylation modification.

scholarly journals TEMPORAL ORDER IN MAMMALIAN CELLS

1969 ◽  
Vol 43 (2) ◽  
pp. 207-219 ◽  
Author(s):  
Robert R. Klevecz
Keyword(s):  
Enzyme Activity ◽  
Mammalian Cells ◽  
Temporal Order ◽  
Actinomycin D ◽  
Chinese Hamster ◽  
Enzyme Synthesis ◽  
S Phase ◽  
Selection System ◽  
Enzyme Protein ◽  
Chinese Hamster Cells

Chinese hamster cells were synchronized by the Colcemid-selection system. In cells with a division cycle time of 11.5–12 hr, the activity of the enzyme lactate dehydrogenase (LDH) underwent marked oscillations with a 3.5-hr period. Precipitation of labeled LDH enzyme with specific antibody indicated that the enzyme activity changes were the result of intermittent enzyme synthesis and relatively constant degradation. Inhibition of normal DNA replication with 4 mM of thymidine, while reducing the amount of new enzyme synthesized, did not prevent oscillations from occurring. Similarly, actinomycin D (AcD) added at the time of synchronization allowed some new enzyme synthesis to proceed in an oscillatory manner. LDH synthesis went on at nearly normal rates when AcD was added in the middle of S phase. However, addition of cycloheximide to cultures at any time in the cycle caused an immediate drop in levels of activity and in enzyme protein. The half-life of LDH, calculated either from loss of enzyme activity or precipitable radioactivity in cycloheximide-treated cultures, was between 2 and 2.5 hr.

The Relevance of Intra-Cellular Damage to Effects in the Complex Organism

1968 ◽  
Vol 70 (2) ◽  
pp. 152-162
Author(s):  
R. H. Mole
Keyword(s):  
Cell Division ◽  
Mammalian Cells ◽  
Radiation Effects ◽  
Specific Activity ◽  
A Priori ◽  
High Specific Activity ◽  
Cellular Damage ◽  
Complex Organism ◽  
Interesting Approach

As the three previous papers in this Symposium have shown (Ord and Stocken 1968; Evans 1968; Court-Brown 1968) the exploration of radiation effects within the cell has its own intrinsic fascination quite apart from its relevance to an understanding of the phenomena which may be observed after irradiation of the complex organism, vertebrate or invertebrate. Whitmore (cf. Whitmore Gulyas and Botond 1965) has been one of the pioneer workers on a previously unsuspected phenomenon, the quantitatively substantial recovery of the ability to divide which occurs within the first hour or two after exposure to low LET radiation. In cultures of mammalian cells in vitro this kind of recovery was shown to have nothing to do with cell division, to occur at all stages of the cell cycle, and to be little, if at all, affected by a reduction in temperature which would be expected a priori to modify profoundly the rate of chemical reactions. Whitmore's technique for synchronising asynchronously dividing cell cultures by the selective suicide of DNA synthesising cells following the incorporation of radioactively labelled DNA precursors with high specific activity has a wide application (Whitmore and Gulyas 1966). Workers from his laboratory have proposed an interesting approach to the real meaning of “ability to divide” and “recovery” based on the idea that there is a certain probability of failure at each cell division and that this probability may be permanently increased by exposure to radiation (Till, McCulloch and Siminovitch 1964). Thus radiation damage may be expressed by failure of cell division not only at the first or second divisions after exposure but also at any subsequent division in the distant future.

scholarly journals Thymidine-requiring mutants of Dictyostelium discoideum.

1984 ◽  
Vol 4 (12) ◽  
pp. 2784-2791 ◽  
Author(s):  
G Podgorski ◽  
R A Deering
Keyword(s):  
Dictyostelium Discoideum ◽  
Nuclear Dna ◽  
Specific Activity ◽  
High Specific Activity ◽  
Normal Growth ◽  
Thymidylate Synthetase ◽  
Synthetase Activity ◽  
Cell Extracts ◽  
Dna Replication And Repair

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.

scholarly journals Inhibition of Thymidylate Synthetase Activity Induced in Varicella-Zoster Virus Infected Cells by (E)-5-(2-bromovinyl)-2′-deoxyuridine

1994 ◽  
Vol 5 (3) ◽  
pp. 191-194 ◽  
Author(s):  
T. Yokota ◽  
K. Konno ◽  
S. Shigeta
Keyword(s):  
Mammalian Cells ◽  
De Novo ◽  
Specific Interaction ◽  
Thymidylate Synthetase ◽  
Synthetase Activity ◽  
Focus Formation ◽  
Varicella Zoster ◽  
Key Enzyme ◽  
Infected Cells ◽  
Viral Form

The authors investigated the effect' of thymidylate synthetase (TS) on the antivariclllazoster virus (VZV) activity of ( E)5-(2-bromovinyl)-2′-deoxyuridine (BVDU). TS catalyses the conversion of deoxyuridylate (dUMP) to thymidylate (dTMP) and is a key enzyme in pyrimidine biosynthesis, providing the only source of dTMP synthesized de novo in mammalian cells. VZV encodes a specialized viral form of TS. TS activity in cells infected with VZV (TIO-VZV and TK−-VZV) increased proportionally with focus formation. From kinetic analysis using the Michaelis-Menten equation, the authors determined a Km value of 6.6μm for dUMP of TS induced in VZV-infected cells and a corresponding value from mock-infected cells of only 2.8μm. BVDU inhibited the induction of TS activity in TK+-VZV-infected cells at concentrations under ×10−3μm, but did not inhibit TS activity of TK−-VZV- or mock-infected cells at concentrations as high as 10μm. Inhibitory activity of BVDU against TS induced in TK+-VZV-infected cells appears to occur when BVDU is phosphorylated to BVDU monophosphate by viral pyrimidine kinase. These results suggest that the selective inhibitory action of BVDU on VZV replication depends on a specific interaction with both viral TK and TS. 5-Bromodeoxyuridine and 5-iododeoxyuridine inhibited TS activities induced in both VZV (TK+, TK−) and mock-infected cells. Other antiherpes compounds [i.e. 1-β-D-arabinofuranosyl- E-5-(2-bromovinyl)uracil, 9- 2-hydroxyethoxymethyl)-guanine, arabinosyladenine, and others] did not inhibit TS activity in VZV-infected cells at concentrations Of 10μm.

scholarly journals Trypanosoma cruzi: modification of alkaline phosphatase activity induced by trypomastigotes in cultured human placental villi

1990 ◽  
Vol 32 (6) ◽  
pp. 403-408 ◽  
Author(s):  
Ricardo E. Fretes ◽  
Sofía P. de Fabro
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Trypanosoma Cruzi ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Mammalian Cells ◽  
Specific Activity ◽  
Human Trophoblast ◽  
Placental Villi

Human term placental villi cultured ''in vitro" were maintained with bloodstream forms of Trypanosoma cruzi during various periods of time. Two different concentrations of the parasite were employed. Controls contained no T. cruzi. The alkaline phosphatase activity was determined in placental villi by electron microscopy and its specific activity in the culture medium by biochemical methods. Results showed that the hemoflagellate produces a significant decrease in enzyme activity as shown by both ultracytochemical and specific activity studies and this activity was lower in cultures with high doses of parasites. The above results indicate that the reduction in enzyme activity coincides with the time of penetration and proliferation of T. cruzi in mammalian cells. These changes may represent an interaction between human trophoblast and T. cruzi.

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