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 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 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 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 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

Abstract 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.

scholarly journals Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69

Catalysts ◽  
2018 ◽  
Vol 8 (8) ◽  
pp. 325 ◽  
Author(s):  
He Chen ◽  
Jie Huang ◽  
Binyun Cao ◽  
Li Chen ◽  
Na Song ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Lactobacillus Plantarum ◽  
Industrial Development ◽  
Extraction Efficiency ◽  
Specific Activity ◽  
Cell Envelope ◽  
Serine Proteinase ◽  
Kinetic Studies ◽  
Predicted Values ◽  
Hydrolyze Whey Protein

Lactobacilli cell-envelope proteinases (CEPs) have been widely used in the development of new streams of blockbuster nutraceuticals because of numerous biopharmaceutical potentials; thus, the development of viable methods for CEP extraction and the improvement of extraction efficiency will promote their full-scale application. In this study, CEP from a novel wild Lactobacillus plantarum LP69 was released from cells by incubating in calcium-free buffer. The extraction conditions of CEP were optimized by response surface methodology with the enzyme activity and specific activity as the detective marker. The optimal extraction conditions were: time of 80 min, temperature of 39 °C and buffer pH of 6.5. Under these conditions, enzyme activity and specific activity were (23.94 ± 0.86) U/mL and (1.37 ± 0.03) U/mg, respectively, which were well matched with the predicted values (22.12 U/mL and 1.36 U/mg). Optimal activity of the crude CEP occurred at pH 8.0 and 40 °C. It is a metallopeptidase, activated by Ca2+, inhibited by Zn2+ and ethylene-diamine-tetra-acetic acid, and a serine proteinase which is inhibited by phenylmethylsulfonyl fluoride. Kinetic studies showed that CEP from LP69 could hydrolyze whey protein, lactoglobulin and casein. Our study improves the extraction efficiency of CEPs from LP69, providing the reference for their industrial development.

scholarly journals Introgression of Drosophila simulans Nuclear Pore Protein 160 in Drosophila melanogaster Alone Does Not Cause Inviability but Does Cause Female Sterility

Genetics ◽  
2010 ◽  
Vol 186 (2) ◽  
pp. 669-676 ◽  
Author(s):  
Kyoichi Sawamura ◽  
Kazunori Maehara ◽  
Shotaro Mashino ◽  
Tatsuo Kagesawa ◽  
Miyuki Kajiwara ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Drosophila Simulans ◽  
Female Sterility ◽  
Nuclear Pore ◽  
Nuclear Pore Protein ◽  
Pore Protein
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