Thymidylate synthetase purified to homogeneity from human leukemic cells.

Abstract

Thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) from a human leukemic cell line has been purified to homogeneity with one-step affinity column chromatography. The purified enzyme has a specific activity of 3.8 jtmo /min per mg of protein, which corresponds to a turnover number of 250. These are the highest values reported for a thymidylate synthetase from neoplastic tissue. A ratio of 1.7 mol of 5-fluoro-2'.deoxyuridylate binds per mol of enzyme in the presence of 5,10-methylenetetrahydrofolate. The ternary complex so formed migrates intact on denaturing gels and can be precipitated with trichloroacetic acid; however, urea dissociates the ternary complex. The human thymidylate synthetase is composed of two subunits of 33,000 daltons each. It contains more residues of cysteine, glycine, and arginine and fewer of histidine than the well-studied thymidylate synthetase from Lactobacillus casei. Thymidylate synthetase (5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) has been the object of considerable interest because of its key function in mammalian DNA synthesis as the only de novo source of thymidylate. This enzyme is believed to be the major site of action of the cancer chemotherapeutic drug 5-fluorouracil due to inhibition by the metabolite 5-fluoro-2'-deoxyuridylate (FdUMP) (1). FdUMP, 5,10-methylenetetrahydrofolate (5,10-CH2H4PteGlu), and thymidylate synthetase form a ternary complex (2), which is joined by covalent bonds in some cases-i.e., is stable to protein denaturants and limited proteolysis (3)(4)(5)(6). In other cases, the ternary complex is dissociated partially (7) (16) and was used without purification. H4MTX was lyophilized in ampules, sealed in N2, and stored in the dark at -25°C.Aminoethyl polyacrylamide gel beads (Aminoethyl Bio-Gel P-150) were purchased from Bio-Rad, as were the reagents for polyacrylamide gel electrophoresis. Sephadex G-100 was obtained from Pharmacia. Other biochemicals were from Sigma.Cell Culture. CCRF-CEM human lymphoblastic leukemia cells (17) were thawed from mycoplasma-free stocks at 21/2-month intervals and were monitored routinely for absence of mycoplasma. These cells were grown in suspension in RPMI 1640 medium (GIBCO) supplemented with 10% fetal calf serum (GIBCO) and were harvested in late logarithmic growth, washed once with phosphate-buffered saline, and used immediately or stored in liquid N2.Affinity Column Preparation. Aminoethyl polyacrylamide (0.5 g) was hydrated with water and reacted with 20 mg of H4MTX and 150 mg of 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide-HCI by the method of Slavik et al. (12), except that N2 gas was used. About 40% of the resultant gel was transferred into a small plastic column (5-ml Pipetman tips partially stuffed with glass wool) in a cold room (40C). The light-tan beads were washed overnight with 200 ml of 2.5% NaHCO3/0.5 M KCl containing 0.15 M 2-mercaptoethanol. The columns were then washed with 100 ml of 10 mM potassium phosphate buffer (pH 7.4) c...

Stable inhibition of human thymidylate synthase expression following retroviral introduction of an siRNA gene

“…13,25 Trichloroacetic acid-insoluble material was collected by filtration through glass fiber filters and analyzed for radioactivity in a scintillation counter. In the indicated experiments, the amount of TS enzyme was normalized to the amount of total protein in the cell extract, which was determined by the DC Protein Assay (BioRad).…”

Mutants of chinese hamster cells deficient in thymidylate synthetase

“…The amount of the TS enzyme in cytosol was determined by measuring the binding of 3H-FdUMP in the presence of 5,10-CHz-FH4 according to the published procedures (Santi et al, 1974a;Lockshin et al, 1979). The number of FdUMP binding sites can be taken as a direct measure of the enzyme level in the cell.…”

A Novel Approach to Cloning and Expression of Human Thymidylate Synthase

“…Phan et al (2001) found that TS functions as an RNA binding protein that regulates the expression of its own mRNA translation, the emergence of resistance to the treatment is often related to the increased level of TS in cancer cells, which have been linked to the elimination of TS binding to its own mRNA upon drug binding. During the early stage, TS was obtained from human cells (Lockshin et al, 1979;Dolnick et al, 1977). With the development of gene recombination and prokaryotic expression system, it is feasible to expression human TS in E. coli.…”