TY - JOUR
T1 - Binding of 5-fluorodeoxyuridylate to thymidylate synthase in human colon adenocarcinoma xenografts
AU - Houghton, J. A.
AU - Torrance, P. M.
AU - Radparvar, S.
AU - Williams, L. G.
AU - Houghton, P. J.
N1 - Funding Information:
This werk was supported by grant CA 32613 from thr National Cancer Institute and by Amrrican Lcbanrsr Syrian Associatcd Charities.
PY - 1986/4
Y1 - 1986/4
N2 - The formation and stability of the covalent ternary complex formed between thymidylate synthase (E.C. 2.1.1.45), 5-fluoro 2′-deoxyuridylate (FdUMP) and 5,10-methylenetetrahydrofolate (CH2-H4PteGlu) has been examined in cytosols derived from xenografts of human colon adenocarcinomas. The rate of association (ka) for FdUMP was low being between 3.4 ± 0.9 and 10.2 ± 2.6 × 106M-1 min-1, with the lowest ka value being determined in cytosols from a tumor (HxELC2) which has demonstrated some sensitivity to 5-fluoropyrimidines. Relative to reported ka values for human leukemic cells, the rate of association of FdUMP was 20- to 59-fold lower. This difference is not a consequence of FdUMP catabolism, or metabolism of CH2-H4PteGlu. In cytosols the apparent Km values for dUMP (3.6-4.2 μM) and [6RS]-CH2-H4PteGlu (25-26.7 μM) were similar to reported values for human enzyme. Data derived from cytosols were similar to those derived using affinity purified enzyme from HxVRC5 colon adenocarcinoma xenografts. The net dissociation of [6-3H] FdUMP from the covalent ternary complex was 31-33 min in the absence of added CH2-H4PteGlu, and the rate of dissociation was dependent upon the concentration of cofactor. The concentration of [6RS]-CH2-H4PteGlu required to stabilize ternary complex derived from HxELC2 cytosols was slightly lower than that required for the same degree of stabilization of complex formed in cytosols from resistant tumors (HxGC3, HxVRC5). Addition of 5-CHO-H4PteGlu, 5-CH3-H4PteGlu, H2PteGlu, and PteGlu did not stabilize the covalent complex, but H4PteGlu substituted for CH2-H4PteGlu.
AB - The formation and stability of the covalent ternary complex formed between thymidylate synthase (E.C. 2.1.1.45), 5-fluoro 2′-deoxyuridylate (FdUMP) and 5,10-methylenetetrahydrofolate (CH2-H4PteGlu) has been examined in cytosols derived from xenografts of human colon adenocarcinomas. The rate of association (ka) for FdUMP was low being between 3.4 ± 0.9 and 10.2 ± 2.6 × 106M-1 min-1, with the lowest ka value being determined in cytosols from a tumor (HxELC2) which has demonstrated some sensitivity to 5-fluoropyrimidines. Relative to reported ka values for human leukemic cells, the rate of association of FdUMP was 20- to 59-fold lower. This difference is not a consequence of FdUMP catabolism, or metabolism of CH2-H4PteGlu. In cytosols the apparent Km values for dUMP (3.6-4.2 μM) and [6RS]-CH2-H4PteGlu (25-26.7 μM) were similar to reported values for human enzyme. Data derived from cytosols were similar to those derived using affinity purified enzyme from HxVRC5 colon adenocarcinoma xenografts. The net dissociation of [6-3H] FdUMP from the covalent ternary complex was 31-33 min in the absence of added CH2-H4PteGlu, and the rate of dissociation was dependent upon the concentration of cofactor. The concentration of [6RS]-CH2-H4PteGlu required to stabilize ternary complex derived from HxELC2 cytosols was slightly lower than that required for the same degree of stabilization of complex formed in cytosols from resistant tumors (HxGC3, HxVRC5). Addition of 5-CHO-H4PteGlu, 5-CH3-H4PteGlu, H2PteGlu, and PteGlu did not stabilize the covalent complex, but H4PteGlu substituted for CH2-H4PteGlu.
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U2 - 10.1016/0277-5379(86)90119-7
DO - 10.1016/0277-5379(86)90119-7
M3 - Article
C2 - 3732354
AN - SCOPUS:0022626114
SN - 0277-5379
VL - 22
SP - 505
EP - 510
JO - European Journal of Cancer and Clinical Oncology
JF - European Journal of Cancer and Clinical Oncology
IS - 4
ER -