Purification and Properties of Thymidylate Synthetase from Pig Thymus

Abstract

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.