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

Slavík, K.; Rode, Wojciech; Slavíková,

doi:10.1021/bi00664a014

Cited by 25 publications

(6 citation statements)

References 22 publications

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“…methotrexate-Sepharose affinity gel and eluting those proteins not specifically bound by using a high-salt wash. Thymidylate synthase from several mammalian and bacteial sources can bind significantly to folate analog-affinity columns only in the presence of dUMP (20)(21)(22). The results with thymidylate synthase and H2folate reductase from C. fasciculata were strikingly different; both enzymes bound to the affinity resin, remained bound through a wash containing 1 M KCI which removed most extraneous proteins, and were eluted together by H2folate (Table 1; Fig.…”

Section: Resultsmentioning

confidence: 99%

Dihydrofolate reductase: thymidylate synthase, a bifunctional polypeptide from Crithidia fasciculata.

Ferone¹,

Roland²

1980

Proc. Natl. Acad. Sci. U.S.A.

109

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Section: Resultsmentioning

confidence: 99%

Dihydrofolate reductase: thymidylate synthase, a bifunctional polypeptide from Crithidia fasciculata.

Ferone¹,

Roland²

1980

Proc. Natl. Acad. Sci. U.S.A.

109

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“…Homogeneous CCRF-CEM thymidylate synthetase has a specific activity (3.8 units/mg of protein) 200 times greater than that reported as the minimum specific activity of thymidylate synthetase derived from human leukemic blast cells (9) and is, in fact, greater than the specific activity of thymidylate synthetase isolated from any mammalian tissue so far reported. Pure CCRF-CEM thymidylate synthetase has a specific activity comparable to the L. casei enzyme (14,29), but lower than that of the enzyme isolated from E. coli (12) or the T2 bacteriophage-induced thymidylate synthetase (7). The turnover number of the enzyme at 37°C is calculated as 250/min.…”

Section: Discussionmentioning

confidence: 99%

“…The turnover number of the enzyme at 37°C is calculated as 250/min. H4MTX acts as a specific affinity ligand for purification of thymidylate synthetase in the presence of dUMP through formation of a tightly bound complex which retains the enzyme on the column in the presence of the high-ionic-strength buffers required to wash out all contaminating proteins (12). When dUMP is eliminated, the enzyme elutes from the column.…”

Section: Discussionmentioning

confidence: 99%

“…Until recently (9), few attempts were made to isolate thymidylate synthetase from human tumors, presumably because of its low levels in human tissues and difficulties in stabilizing the mammalian enzyme (10)(11)(12) (14,15). H4MTX was prepared by catalytic hydrogenation of methothrexate (16) and was used without purification.…”

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confidence: 99%

“…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).…”

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confidence: 99%

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Thymidylate synthetase purified to homogeneity from human leukemic cells.

Lockshin¹,

Moran²,

Danenberg³

1979

Proc. Natl. Acad. Sci. U.S.A.

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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...

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