TY - JOUR
T1 - Improved measurement of thymidylate synthetase activity by a modified tritium-release assay
AU - Armstrong, R. Douglas
AU - Diasio, Robert B.
N1 - Funding Information:
This study was supported by Public Health Service Research Grant CA-23412 awarded by the National Cancer Institute, DHEW.
PY - 1982/6
Y1 - 1982/6
N2 - Accurate quantitation of thymidylate synthetase activity using a tritium-release assay is dependent upon measurement of only that tritium released from deoxy[5-3H]uridine monophosphate ([3H]dUMP) during the biosynthesis of thymidylate. Removal of remaining [3H]dUMP on completion of the assay by charcoal adsorption and correction for the nonenzymatic release of tritium are necessary. Although over 99% of [3H]dUMP is removed immediately following addition of charcoal, these studies demonstrate that sufficient [3H]dUMP can remain to prevent accurate measurement of low levels of thymidylate synthetase activity. By delaying measurement of radioactivity for at least 24 h following addition of charcoal, this problem is minimized. To account for nonenzymatic release of tritium, a blank containing enzyme extract with omission of ±,l-5,10-methylenetetrahydrofolate is demonstrated to be more effective than the commonly used blank in which water is substituted for enzyme extract. In samples containing 5-fluoro-2′-deoxyuridine monophosphate (FdUMP), a potent inhibitor of thymidylate synthetase activity, an alternative blank containing a high concentration of FdUMP (approximately 1mM) is useful in demonstrating a theoretical maximal or complete inhibition of thymidylate synthetase activity.
AB - Accurate quantitation of thymidylate synthetase activity using a tritium-release assay is dependent upon measurement of only that tritium released from deoxy[5-3H]uridine monophosphate ([3H]dUMP) during the biosynthesis of thymidylate. Removal of remaining [3H]dUMP on completion of the assay by charcoal adsorption and correction for the nonenzymatic release of tritium are necessary. Although over 99% of [3H]dUMP is removed immediately following addition of charcoal, these studies demonstrate that sufficient [3H]dUMP can remain to prevent accurate measurement of low levels of thymidylate synthetase activity. By delaying measurement of radioactivity for at least 24 h following addition of charcoal, this problem is minimized. To account for nonenzymatic release of tritium, a blank containing enzyme extract with omission of ±,l-5,10-methylenetetrahydrofolate is demonstrated to be more effective than the commonly used blank in which water is substituted for enzyme extract. In samples containing 5-fluoro-2′-deoxyuridine monophosphate (FdUMP), a potent inhibitor of thymidylate synthetase activity, an alternative blank containing a high concentration of FdUMP (approximately 1mM) is useful in demonstrating a theoretical maximal or complete inhibition of thymidylate synthetase activity.
KW - 5-fluorouracil
KW - thymidylate synthetase
KW - tritium-release assay
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U2 - 10.1016/0165-022X(82)90060-4
DO - 10.1016/0165-022X(82)90060-4
M3 - Article
C2 - 7108126
AN - SCOPUS:0020315641
SN - 0165-022X
VL - 6
SP - 141
EP - 147
JO - Journal of Biochemical and Biophysical Methods
JF - Journal of Biochemical and Biophysical Methods
IS - 2
ER -