Purification and Properties of Thymidylate Synthetase from Pig Thymus

Abstract

GVPTA, V. S., and MELDRUM, J. B. Purification and properties sf thymidylate synthetase frsm pig thymus.Can. J. Biochem. 9,352-362 (1972).Thymidylate synthetase of pig thymus has been separated into two principal forms (designated I and 11, based on their order of elution) by chromatography on CM-Sephadex. By the use of (NH,),SB, the synthetase activity was separated into two fractions, and these were further purified by gel filtration using Sephadex G-IBQ and chromatography on CM-Sephadex. The highest specific activity obtained for I and I1 was 10.4 and 14.3 p o l of thymidine-5'-phosphate per hour per milligram of protein at 25" and pH 7.3 which represents a purification of 1688and 2630-fold, respectively. EBectrophoretically, I and II appear to be 78-88% pure. The Michaelis constants of 7.4 x 10s" M, 1.7 x M, and 1.8 x los4 M for HI with respect to deoxyuridine-5'-phosphate, 5,1O-methlenetetrahydrofolate, and uridine-5'-phosphate, respectively, have been determined. A double pH optima in the r a n g sf 4.4-4.8 and 7.2-7.4 in 2-N-wsrpholinoethane suBfonic acid buffer was exhibited by both forms. F o m s I and II showed maximal catalytic activity only in the presence of sulfkydryl compounds (a mM) and also had the ability to methylate uridine-5'-phosphate, although at a dower rate (ca. 28% and 13"/,, respectively) compared with the rase s f methylation of deoxyuridine-5'phosphate. Both deoxyuridine-5'-phosphate and tetrahydrofolate (to a lesser extent) aEorded protection to I1 against heat inactivation. GUPTA, V. S., et MELDRUM, J. B. Purification and properties of thymidylate synthetase frsm pig thymus. Can. J. Biochem. 5% 352-362 (1972). La thymidylixte synthktase de thymus de porc est dparie en deux fomes principales (appelees I et I1 selon B'ordre de leur elution) par chromatographie sulr CM-Skphadex. L'emploi du QNH,),S04 pemet de stparer la synthktase en deux fractions que i90n a purifikes par filtration sur gel de Stphadex G-IBKB et par chromatographie sur CM-Sephadex. L'activitk spkcifique H a plus &levee obtenue avec I et II est de 10.4 et 16.3 pmol de thymidine-5'-phosphate gar heure par mg de protkine A 25" et i+ pH 7.3, ce qui reprtsente Bane purification de I)ordre de 1688 et 2636) fois resptivement. EBectrophoretiquernent, I et I1 seraient de 70 B 80 pour-cent pures. Les constantes de Michaelis de B a forme IH, mesurees avec le dksoxyuridine-5'-phosphate, B e 5,10-rnkthyl+netetrahydrofohte ct l'uridine-5'-phosphate, sont resptivement de 7.4 x M, 1.7 x BIB-5M et 1.8 x la-" M. Dans 1e tampon MES, les deux forms ont un double pH optimum: I'un entre 6.6 et 6.8 et l'autre entre 7.2 et 7.4. L'activitt catalytique de I et I1 est maximum seu%ernent en prksence de composes sulfbydryles (60 m M ) et toutes deux poss6dent le porevoir de mkthyier 1'uridine-5'-phosphate, mais a une vitesse plus lente (ca. 28% et 13% resydectivement) cornpark I celle de la methylation du desoxyuridine-5'phosphate. Le dboxyuridine-5'-phosphate et le tttrahydrofolate ( B un degrt moindre) protegent tous deux la forme HI mntae l'ina...

Thymidylate Synthetase

“…Similar thiol activation has been reported for thymidylate synthetase from chick embryos (20), dichloromethotrexate-resistant L. casei (22), Ehrlich ascites carcinoma cells (31), and pig thymus (32). All of these studies support the concept that a thiol group is essential for catalysis.…”

Thymidylate Synthase Activity from Chlamydomonas Cells and Cultured Tissues of Nicotiana, Pinus, and Daucus

“…The synthesis of TMP via the methylation ofdUMP is catalyzed by thymidylate synthase (methylene tetrahydrofolate:deoxyuridine-5'-monophosphate C-methyltransferase, EC 2.1.1.6), which has been studied extensively in certain bacterial and animal systems (6)(7)(8)(9). There is a paucity of information on the direct measurement of thymidylate synthase from plants; hence, the presence of the enzyme is predicated largely on indirect observations such as an altered mitotic index (16) and decreased DNA synthesis and content (19) after treatment with 5-FUdR, a potent inhibitor of thymidylate synthase when phosphorylated (8).…”

“…First, we have found that the published assay procedures are neither sufficiently sensitive nor, in the case of spectrophotometric approaches, specific when plant extracts are used. Second, normal plant tissues, as in mammalian tissues (6,9), would contain very small amounts of thymidylate synthase unless they were about to initiate DNA synthesis (14). Consequently, studies of thymidylate synthase in bacteria and animals have focused on systems undergoing rapid cell proliferation (5,8,9,11,14 (22) were grown in nipple flasks with 2.0 ,ug/g naphthalene acetic acid and 1% coconut milk (18).…”

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

“…Provided that adequate control experiments are performed (as discussed by Chan, 1976), it appears that the study of subunits immobilized on Sepharose can provide information regarding the effects of alterations in quaternary structure on protein function. Blakley , 1963;Lorenson et al, 1967;Crusberg et al, 1970;Leary and Kisliuk, 1971; Gupta and Meldrum, 1972;Galivan et al" 1974). Affinity chromatography methods using Sepharose bound with either deoxyuridylate (Danenberg et al, 1972) or 5-fluorodeoxyuridylate (Whiteley et al, 1974)-a strong inhibitor of the enzyme forming a covalent complex with it-were used successfully for purification of thymidylate synthetase from L. casei resistant to dichloromethotrexate, which is an exceptionally rich source of the enzyme (Crusberg and Kisliuk, 1969;Dunlap et al, 1971).…”