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Biomedical subjects

W Rode

Publications and source records attributed to W Rode.

46 records · Page 3Linked to original sources
PubMed1983

Altered thymidylate synthetase in 5-fluorodeoxyuridine-resistant Ehrlich ascites carcinoma cells.

Thymidylate synthetase from 5-fluorodeoxyuridine-resistant Ehrlich ascites carcinoma cells was purified to a state close to electrophoretical homogeneity (sp. act. = 1.3 mumoles/min/mg protein) and studied in parallel with the homogeneous preparation of the enzyme from the parental Ehrlich ascites carcinoma cells. The enzyme from the resistant cells compared to that from the parental cells showed: (i) a higher turnover number (at least 91 against 31 min-1), (ii) a higher inhibition constant (19 against 1.9 nM) for FdUMP (a tight-binding inhibitor of both enzymes), (iii) a lower activation energy at temps above 36 degrees (1.37 against 2.59 kcal/mole), and (iv) a lower inhibition constant (26 against 108 microM) for dTMP, inhibiting both enzymes competitively vs dUMP.

Animals↗
PubMed1982

Properties of thymidylate synthetase from Ehrlich ascites carcinoma cells. Effect of Mg2/ and MgATP2-.

Ehrlich ascites carcinoma thymidylate synthetase was purified to electrophoretic homogeneity by affinity chromatography on 10-formyl-5,8-dideazofolate-ethyl-Sepharose. Electrophoretic analysis of the formation of the enzyme-5-fluorodeoxyuridylate-5,10-methylenetetrahydrofolate complexes showed the presence of two binding sites for 5-fluorodeoxyuridylate on the enzyme molecule. Molecular weight of the native enzyme was found to be 78,5000, whereas that of its monomer was 38, 500. The apparent Michaelis constants for dUMP and (+/-)-L-5,10-methylenetetrahydrofolate were 1.3 +/- 0.4 and 32.2 +/- 0.7 micrometers respectively. Phosphate acted as a weak inhibitor, competitive toward dUMP. The enzyme reaction exhibited a temperature-dependent change of activation energy, reflected in the binding affinity of dUMP, with a transitional temperature of 35.8 degrees. Both Mg2+ and MgATP2- were strong activators of the enzyme, MgATP2- being more effective.

Adenosine Triphosphate↗
PubMed1980

Regulation of thymidylate synthetase in mouse leukemia cells (L1210).

L1210 mouse leukemia cells were synchronized by isoleucine deprivation followed by hydroxyurea treatment. Changes of thymidylate synthetase activity both in cell extracts and in the intact cells (in situ) were followed throughout the cell cycle. The enzyme activity assayed in situ, by measuring tritium released from [5-3H]deoxyuridine, changed markedly in relation to the DNA synthesis pattern, whereas that assayed in cell extracts remained unchanged. In the S phase of the cell cycle, activity of the enzyme was much higher as measured in situ than as measured in cell extracts. Hydroxyurea (1 mM) inhibited DNA synthesis in exponentially growing L1210 cells and was found to inhibit thymidylate synthetase activity in situ. However, the enzyme activity in cell extracts was not inhibited by the drug. In cells with DNA synthesis inhibited by hydroxyurea, the pattern of change of in situ activity with time suggested product inhibition. Results of isotope-dilution analysis of the release of tritium from [5-3H]deoxyuridine in the intact cells indicated that thymidine added to the medium caused strong inhibition of thymidylate synthetase, the most likely limiting step in this pathway. These data allow the suggestion that, during cell growth, thymidylate synthetase activity is regulated by thymine nucleotide(s), and is dependent on the rate of DNA synthesis as well as the availability of the salvage pathway substrate, thymidine.

Animals↗
PubMed1979

Purification of mammalian tumor (L1210) thymidylate synthetase by affinity chromatography on stable biospecific adsorbent. Stabilization of the enzyme with neutral detergents.

Thymidylate synthetase from mouse leukemic L1210 cells was purified to electrophoretic homogeneity with 70% yield as a result of an affinity chromatography procedure based on reversible deoxyuridylate-dependent binding of the enzyme to a stable biospecific adsorbent, 10-formyl-5,8-dideazafolate, immobilized on aminoethyl-Sepharose. The presence of neutral detergents, Triton X-100, or Nonidet P40 stabilized thymidylate synthetase during purification. Analytical electrophoresis of the enzyme treated with an excess of 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate showed the presence of two forms of thymidylate synthetase--5-fluorodeoxyuridylate.5,10-methylenetetrahydrofolate complex, indicating that there are two binding sites for 5-fluorodeoxyuridylate present on the enzyme molecule. Molecular weight of native thymidylate synthetase was found to be 75,000, whereas that for the monomer was 38,500.

Animals↗
PubMed1976

Purification of thymidylate synthetase from enzyme-poor sources by affinity chromatography.

The adsorption of thymidylate synthetase from Escherichia coli B to aminoalkyl-Sepharose with the increasing length of carbon chain (2--6 carbon atoms) was investigated. A correlation was found between the chain length and adsorption effectiveness, increasing from the two- to the six-carbon chain. A hydrophobic chromatography of the enzyme on aminobutyl-Sepharose gave about 20-fold purification. A new affinity chromatography carrier was synthesized containing tetrahydromethotrexate linked to aminoethyl-Sepharose via its carboxylic groups. The carrier adsorbed the enzyme from the crude preparation only in the presence of deoxyuridine 5'-monophosphate (dUMP) in a concentration of 2 X 10(-5) M. The specifically adsorbed thymidylate synthetase was eluted with sacharose-containing buffers in which dUMP was omitted. The purification procedure was applied to a crude thymidylate synthetase preparation from resting E. coli, calf thymus, Sarcoma 180, and Gardner lymphosarcoma. The purified enzyme from all mentioned sources showed one protein band on disc electrophoresis corresponding to enzymatic activity. The formation of a reversible noncovalent complex enzyme-tetrahydromethotrexate-dUMP on the affinity column is supposed.

Ammonium Sulfate↗