TY - JOUR
T1 - Determination of thymidylate synthase activity in colon tumor tissues after treatment with 5-fluorouracil
AU - Houghton, Janet A.
AU - Radparvar, Saeed
AU - Torrance, Pamela M.
AU - Williams, Larry G.
AU - Houghton, Peter J.
N1 - Funding Information:
* This work was supported by award CA 32613 from the National Cancer Institute, and by the American Lebanese Syrian Associated Charities. t To whom correspondence should be addressed. $ Abbreviations: FUra, 5-fluorouracil; [6RS]-CH2-H,PteGlu, racemic mixture of the natural [6R] and unnatural [6S] diastereoisomers of 5,10-methylenetetrahydro-pteroylmonoglutamate; and 10% charcoal suspension, 10% activated charcoal + 0.5% bovine serum albumin + 0.05% dextran.
PY - 1987/4/15
Y1 - 1987/4/15
N2 - The formation and isolation of [6-3H]FdUMP-thymidylate synthase-5,10-methylenetetra-hydrofolate covalent complex have been examined in tumor cytosols incubated with albumin-dextran coated charcoal used to remove endogenous nucleotide. Charcoal suspension (10% charcoal, 0.5% albumin, 0.05% dextran) adsorbed < 98% of dUMP added to cytosols, but it reduced by 42-87% covalent complex isolated from subsequent incubation with [6-3H]FdUMP and cofactor using cytosols from different tumors. Initial treatment of ternary complex with charcoal suspension did not cause a decrease in stability of covalent complex during subsequent incubation (37°), but complex separated from free ligand by 10% charcoal suspension was not stable to further treatment with 4% charcoal suspension. Treatment of tumor cytosols with 10% charcoal suspension, to remove nucleotide, did not decrease the rate at which enzyme catalyzed the release of 3H2O from [5-3H]dUMP, or release active enzyme from the ternary complex. Based on these observations, a sensitive procedure for determining thymidylate synthase activity has been developed in which unbound nucleotides (dUMP, FdUMP) are removed prior to assay of enzyme activity. The procedure is suitable for assay of small samples of tissue or of tissues with a low (or inhibited) level of thymidylate synthase activity.
AB - The formation and isolation of [6-3H]FdUMP-thymidylate synthase-5,10-methylenetetra-hydrofolate covalent complex have been examined in tumor cytosols incubated with albumin-dextran coated charcoal used to remove endogenous nucleotide. Charcoal suspension (10% charcoal, 0.5% albumin, 0.05% dextran) adsorbed < 98% of dUMP added to cytosols, but it reduced by 42-87% covalent complex isolated from subsequent incubation with [6-3H]FdUMP and cofactor using cytosols from different tumors. Initial treatment of ternary complex with charcoal suspension did not cause a decrease in stability of covalent complex during subsequent incubation (37°), but complex separated from free ligand by 10% charcoal suspension was not stable to further treatment with 4% charcoal suspension. Treatment of tumor cytosols with 10% charcoal suspension, to remove nucleotide, did not decrease the rate at which enzyme catalyzed the release of 3H2O from [5-3H]dUMP, or release active enzyme from the ternary complex. Based on these observations, a sensitive procedure for determining thymidylate synthase activity has been developed in which unbound nucleotides (dUMP, FdUMP) are removed prior to assay of enzyme activity. The procedure is suitable for assay of small samples of tissue or of tissues with a low (or inhibited) level of thymidylate synthase activity.
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U2 - 10.1016/0006-2952(87)90083-9
DO - 10.1016/0006-2952(87)90083-9
M3 - Article
C2 - 3593415
AN - SCOPUS:0023187994
SN - 0006-2952
VL - 36
SP - 1285
EP - 1289
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 8
ER -