Abstract
Publisher Summary This chapter describes the purification procedure of thymidylate synthetase (TMP synthetase) enzyme from Lactobacillus casei organism. The methylenetetrahydrofolate-dependent reductive methylation of deoxyuridine monophosphate (dUMP) is followed spectrophotometrically at 340 nm by observing the formation of 7, 8-dihydrofolate. Enzyme activity is measured by monitoring the progress of the reaction through the loss of tritium to the aqueous solvent from [5- 3 H]dUMP or by the use of a filter assay that traps the 5-fluorodUMP-methylenetetrahydrofolatethymidylate synthetase ternary complex. An amethopterin-resistant strain of Lactobacillus casei is obtained as described by culturing Lactobaciilus casei var. rhamnosus (ATCC 7469) in the presence of the increasing levels of amethopterin until the bacteria grew readily in media containing a drug concentration of 1 × 10 -5 M. Native thymidylate synthetase has a molecular weight of approximately 70,000 based on analytical polyacrylamide gel electrophoresis and gel filtration chromatography. The enzyme is stable over a period of months when stored at 0–5 ° in the presence of 10 mM 2-mercaptoethanol in capped vessels layered with argon.
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