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Abstract
The binding of 5-fluorodeoxyuridylate (FdUMP) to carboxypeptidase-inactivated thymidylate synthase obtained from methotrexate-resistant Lactobacillus casei was investigated using [3H]FdUMP in a trichloroacetic acid precipitation assay and by 19F nuclear magnetic resonance spectroscopy. The cleavage of 1 valine residue from the carboxyl terminus of one of the identical subunits of the enzyme dimer correlates with complete loss of thymidylate synthesis (Aull, J. L., Loeble, R. B., and Dunlap, R. B. (1974) J. Biol. Chem. 249, 1167-1172). We have further investigated the phenomenon of carboxypeptidase A-dependent inactivation of thymidylate synthase by employing immobilized carboxypeptidase A in order to facilitate the isolation and characterization of the inactivated enzyme. The time course of carboxypeptidase treatment of thymidylate synthase has been profiled by the spectrophotometric assay, tritium release assay, trichloroacetic acid precipitation assay (covalent adduct analysis), 19F nuclear magnetic resonance spectroscopy, and amino acid analysis. The techniques utilized in this study yielded results which showed that the completely inactivated enzyme (failure to catalyze thymidylate formation) continued to catalyze both covalent FdUMP-enzyme interactions and the formation of the covalent inhibitory ternary complex with the cofactor, 5,1O-methylenetetrahydrofolate, although to a reduced extent, thus effectively uncoupling these processes from thymidylate synthesis activity.
Highlights
Peutic agents in the clinic as well as thespecific utilization of FdUMP as a valuable research tool in elucidating the mechanism of action of this importantenzyme
Characterization of the Course of Inactivation of Thymidylate Synthase by Immobilized CarboxypeptidaseA-While previous studies of the inactivation of T S by carboxypeptidase A (CPA) employed the soluble form of the exopeptidase, we used immobilized CPA in order to permit the facile isolation of large quantities of CPA-treated thymidylate synthase for further characterization
The correlation of the loss of 1 mol of valine/ enzyme dimer with complete inactivation of thymidylate synthase was established by Aull et al [6], we wanted to verify that theimmobilized carboxypeptidase utilized in the current investigations behaved to the soluble enzyme used in the earlier studies
Summary
The racemic mixture of tion of thymidylate synthase in about 55 minand provided sufficient (+) CHPH4folatewas obtained by adding a 25-fold excess of HCHO enzyme for analysis of activity by the spectrophotometer and tritium to ( 5 ) H4folate prepared from the catalytic hydrogenation of folic release assays, of covalent nucleotide binding by binary and ternary acid in glacial acetic acid as described by Hatefi et al [15]. Aliquots (0.100 and 0.200 ml) were loaded in and the enzyme were prepared by mixing about 0.2 nmol of thymidseparateruns on a Beckman model119C Amino Acid Analyzer ylate synthase with a 50-fold excess of FdUMP in 100 mM acetate equipped with an AA-20 cationic exchange resin. Complex formation in the presence of H4folatewas carried out for 15 min a t 4 'C
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