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.

Multiple Forms of Phosphoglyceromutase from Chicken Breast Muscle

1972 ◽  
Vol 50 (10) ◽  
pp. 1132-1142 ◽  
Author(s):  
Eric James ◽  
R. O. Hurst ◽  
T. G. Flynn
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Breast Muscle ◽  
Heat Inactivation ◽  
Chicken Breast ◽  
Multiple Forms ◽  
Active Components ◽  
Net Charges

Phosphoglyceromutase (2,3-diphospho-D-glycerate: 2-phospho-D-glycerate phosphotransferase, EC 2.7.5.3) has been purified from both frozen and fresh chicken breast muscle. During purification it was found that substrate, 3-phospho-D-glycerate stabilized the enzyme against heat inactivation to almost the same extent as did the cofactor 2,3-diphospho-D-glycerate.Phosphoglyceromutase prepared from frozen chicken breast muscle separated into three peaks of activity (I, II, and III) following chromatography on DEAE-Sephadex in 0.05 μ phosphate buffer, pH 8.0, using a 0.0–0.4 M NaCl gradient. Each peak of activity was shown by polyacrylamide disc gel electrophoresis at pH 9.3 to contain two enzymically active components (isoenzymes Ia Ib, IIa IIb, and IIIa IIIb). Isoenzymes in the same peak had the same specific activity. Phosphoglyceromutase prepared from fresh chicken breast muscle yielded only one peak of activity following chromatography on DEAE-Sephadex. This peak contained two enzymically active components corresponding to isoenzymes Ia and Ib. Additional peaks of activity were not produced when phosphoglyceromutase from fresh muscle was subjected to freezing and thawing.Isoenzyme Ia and mixtures of Ia and Ib, IIa and IIb, and IIIa and IIIb were homogeneous in the ultra-centrifuge sedimenting as single peaks. The sedimentation coefficient obtained for isoenzyme Ia and for Ia and Ib combined was 4.15 S, the diffusion constant 6.62 × 10−7 cm2/s, and the molecular weight calculated from both gel filtration and sedimentation data was of the order of 59 000. These results were confirmed by charge isomer studies which also showed that the isoenzymes of phosphoglyceromutase from frozen chicken breast muscle were proteins of the same size but different net charges.

Comparative studies of activity and properties of ferredoxin–NADP+ reductase during cold hardening of wheat

1976 ◽  
Vol 54 (16) ◽  
pp. 1896-1902 ◽  
Author(s):  
Joseph Riov ◽  
Gregory N. Brown
Keyword(s):  
Activation Energy ◽  
Spring Wheat ◽  
Comparative Studies ◽  
Low Temperatures ◽  
Specific Activity ◽  
Cold Hardening ◽  
Heat Inactivation ◽  
Enzyme Molecule ◽  
Enzyme Specific Activity ◽  
Michaelis Constants

Activity and properties of chloroplast ferredoxin–NADP− reductase (EC 1.6.7.1) were studied during cold hardening of two varieties of wheat (Triticum aestivnm), hardy Kharkov (winter wheat) and much less hardy Rescue (spring wheat), to determine whether adaptation to low temperatures involves changes in the activity and properties of this enzyme. Specific activity of ferredoxin–NADP− reductase increased during hardening of both varieties, but the increase was much greater in the more hardy variety, Kharkov 22 MC. No changes were found in the Michaelis constants for NADPH and 2,6-dichlorophenol indophenol, activation energy values, inhibition constants for p-chloromercuriphenylsulfonate, and sensitivity toward cold and heat inactivation of the enzyme from control and cold-hardened seedlings of both varieties. The data suggest that there is a preferential synthesis of ferredoxin–NADP− reductase during hardening of wheat, but the enzyme molecule remains unchanged.

scholarly journals Purification and properties of uroporphyrinogen III synthase (co-synthase) from an overproducing recombinant strain of Escherichia coli K-12

1989 ◽  
Vol 264 (2) ◽  
pp. 397-402 ◽  
Author(s):  
A F Alwan ◽  
B I A Mgbeje ◽  
P M Jordan
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Heat Inactivation ◽  
Gene Encoding ◽  
Ph Optimum ◽  
Nitrobenzoic Acid ◽  
Purification And Properties ◽  
Lower Reactivity ◽  
In The Wild ◽  
K 12

The Escherichia coli hemD gene, encoding the enzyme uroporphyrinogen III synthase (co-synthase), was cloned into multi-copy plasmids in E. coli cells that were used to generate strains producing up to 1000 times the concentration of the synthase in the wild-type. The enzyme was purified to homogeneity from these strains in milligram amounts. The enzyme is a monomer of Mr 28,000 with an isoelectric point of 5.2 and a pH optimum of 7.8. The specific activity of the purified synthase is 1500 units/mg and the Km for the substrate, pre-uroporphyrinogen, is 5 microM. The N-terminal sequence of the enzyme is Ser-Ile-Leu-Val-Thr-Arg-Pro-Ser-Pro-Ala-Gly-, in agreement with the gene-derived protein sequence. The enzyme contains four 5,5′-dithiobis-(2-nitrobenzoic acid)-titratable groups, one reacting rapidly with the reagent and three further groups having lower reactivity. The enzyme is heat-sensitive, and during heat inactivation all four thiol groups become equally available for reaction.

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.

Purification and Properties of a Neutral Protease from Soil Bacterium WM 122

1977 ◽  
Vol 37 (03) ◽  
pp. 556-565 ◽  
Author(s):  
S. E Papaioannou ◽  
W. J Marsheck
Keyword(s):  
Methyl Ester ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Soil Bacterium ◽  
Ester Hydrochloride ◽  
Apparent Homogeneity ◽  
Purification And Properties ◽  
Hydrolysis Of

SummaryAn extracellular protease SN 687, secreted by the soil bacterium isolate WM 122, has been purified by means of gel filtration, ammonium sulfate precipitation, DEAE-Sephadex and hydroxylapatite chromatography. Apparent homogeneity was ascertained by Polyacrylamide gel electrophoresis. The protease was inactivated by ethylenediamine tetracetic acid (EDTA) but not by diisopropylfluorophosphate (DFP), and it was partially inhibited by serum inhibitors. SN 687 was shown to be of high specific activity against casein and fibrin, but it did not hydrolyze L- lysine -methyl ester dihydrochloride (LME), p-tosyl-L-arginine-methyl ester hydrochloride (TAME) and N-benzoyl-L-tyrosine-ethyl ester hydrochloride (BTEE) synthetic substrates. The optimum pH for hydrolysis of casein was 7.5 and the molecular weight, as determined by gel filtration, was 31,000.

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 Purification and properties of 3-hexulose phosphate synthase and phospho-3-hexuloisomerase from Methylococcus capsulatus

1974 ◽  
Vol 144 (3) ◽  
pp. 477-486 ◽  
Author(s):  
T Ferenci ◽  
T Strøm ◽  
J R Quayle
Keyword(s):  
Equilibrium Constants ◽  
Gel Filtration ◽  
Reverse Reaction ◽  
Methylococcus Capsulatus ◽  
Forward Reaction ◽  
Bivalent Cation ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Michaelis Constants ◽  
Phosphate Synthase

3-Hexulose phosphate synthase and phospho-3-hexuloisomerase were purified 40- and 150-fold respectively from methane-grown Methylococcus capsulatus. The molecular weights of the enzymes were approximately 310000 and 67000 respectively, as determined by gel filtration. Dissociation of 3-hexulose phosphate synthase into subunits of molecular weight approx. 49000 under conditions of low pH or low ionic strength was observed. Within the range of compounds tested, 3-hexulose phosphate synthase is specific for formaldehyde and d-ribulose 5-phosphate (forward reaction) and d-arabino-3-hexulose 6-phosphate (reverse reaction), and phospho-3-hexuloisomerase is specific for d-arabino-3-hexulose 6-phosphate (forward reaction) and d-fructose 6-phosphate (reverse reaction). A bivalent cation is essential for activity and stability of 3-hexulose phosphate synthase; phospho-3-hexuloisomerase is inhibited by many bivalent cations. The pH optima of the two enzymes are 7.0 and 8.3 respectively and the equilibrium constants are 4.0×10-5m and 1.9×102m respectively. The apparent Michaelis constants for 3-hexulose phosphate synthase are: d-ribulose 5-phosphate, 8.3×10-5m; formaldehyde, 4.9×10-4m; d-arabino-3-hexulose 6-phosphate, 7.5×10-5m. The apparent Michaelis constants for phospho-3-hexuloisomerase are: d-arabino-3-hexulose 6-phosphate, 1.0×10-4m; d-fructose 6-phosphate, 1.1×10-3m.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland

1975 ◽  
Vol 151 (2) ◽  
pp. 263-270 ◽  
Author(s):  
S A Betts ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Phosphogluconate Dehydrogenase ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Final Purification Step

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.

scholarly journals PREPARATION, PURIFICATION AND PROPERTIES OF LIPASE FROM HEPATOPANCREAS OF TRA (PANGASIUS) CATFISH

2011 ◽  
Vol 14 (3) ◽  
pp. 5-11
Author(s):  
Thy Bao Vuong ◽  
Lam Bich Tran ◽  
Duan Luu
Keyword(s):  
Heavy Metals ◽  
Molecular Weight ◽  
Enzyme Activity ◽  
Crude Extract ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Purification And Properties ◽  
Temperature Optima

Lipase from the hepatopancreas of Tra (Pangasius) catfish was purified by ammonium sulfate fractionation, followed by ion-exhange chromatography on DEAE Cellulose and gel filtration Sephadex G-75. The preparation was homogeneous on polyacrylamide disc gel electrophoresis. The specific activity of the purified enzyme was 37.95 times higher than that of the crude extract. The enzyme showed a molecular weight of 57000 Da. The pH and temperature optima of purified lipase were 8 and 500C respectively. Enzyme activity was enhanced by Ca2+ but inhibited by heavy metals Zn2+, Cd2+, Mg2+.

AMP metabolism by the marine bacterium Vibrio (Beneckea) natriegens: purification and properties of adenylate kinase

1981 ◽  
Vol 27 (10) ◽  
pp. 1053-1059 ◽  
Author(s):  
Karamchand Ramotar ◽  
Michael A. Pickard
Keyword(s):  
Molecular Weight ◽  
Adenylate Kinase ◽  
Gel Electrophoresis ◽  
Marine Bacterium ◽  
Specific Activity ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Atp Formation ◽  
Purification And Properties ◽  
Beneckea Natriegens

Adenylate kinase (EC 2.7.4.3) has been purified 484-fold from extracts of Vibrio natriegens to a specific activity of 1350 μmol ADP formed∙min−1∙mg protein−1. The preparation was 97% pure as judged by gel electrophoresis and exhibited molecular weight values of 29 000 by gel filtration and 32 000 by SDS–gel electrophoresis. The isoelectric point was at pH 4.7. Only ATP (Km 0.067 mM), ADP (Km 0.45 mM), and AMP (Km 0.12 mM) exhibited high activity as substrates, though dATP or dAMP could serve as cosubstrates with AMP or ATP, respectively, at reduced rates. The equilibrium constant in the direction of ATP formation was 1.09, and the pH optimum in both directions was broad, from pH 7.2 to pH 7.6. Enzyme activity was sensitive to the thiolalkylating agents iodacetamide and p-chloromercuriphenyl sulfonate.

Sign in / Sign up

Export Citation Format

Share Document