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A Lockshin

Publications and source records attributed to A Lockshin.

30 records · Page 2Linked to original sources
PubMed1982

Thymidylate synthetase - substrate complex formation.

The complexes that thymidylate synthetase (TSase) forms with various potent inhibitors have been intensively studied and thoroughly reviewed. Of particular significance is the covalent ternary complex of TSase-FdUMP-5,10-CH2H4PteGlu. FdUMP is the active metabolite of the widely used anti-cancer drug 5-fluorouracil. This complex is thought to be analogous to a steady-state intermediate of the normal enzyme reaction with the substrate dUMP. In this review, we examine the properties of TSase-dUMP complexes in order to determine if there is an experimental basis for drawing a close analogy between dUMP and FdUMP in their interaction with TSase, and also to evaluate data indicating a potential chemotherapeutic value for TSase-dUMP complexes formed in the presence of folate analogs.

Binding, Competitive↗
PubMed1982

Tight-binding complexes of thymidylate synthetase, folate analogs and deoxyribonucleotides.

Tightly-bound ternary complexes are formed when dTMP synthetase is incubated with deoxyribonucleotides such as FdUMP, dUMP, and dTMP, and folate analogs, presumably mediated by conformational changes in the enzyme induced by these ligands. The structure of both the folate analog and the nucleotide determines the tightness of binding in the ternary complex. For example, the first order rate constant for dissociation of FdUMP from the dTMP synthetase-FdUMP PteGlu complex is almost 100-fold larger than from the dTMP synthetase-FdUMP-PteGlu3 complex. The latter had a slower rate of dissociation than did the covalent dTMP synthetase-FdUMP-5,10-CH4H4Pte- Glu complex, which had been shown to have a Kd of 10(-11) M. FdUMP formed tighter complexes than did either dUMP or dTMP when using PteGlu3, PteGlu, or H2PteGlu as the folate components, but with 5,8-deazafolate complexes formed with all three nucleotides appeared to have about the same stability. H2PteGlu also induced the tight binding of dUMP to dTMP synthetase. A Scatchard plot indicated positive cooperativity in the binding of dUMP to both human and bacterial enzyme in the presence of H2PteGlu. These results suggest the possibility that when cells are exposed to MTX the decrease in dTMP synthetase activity may be due largely to formation of inhibitory enzyme-dUMP-H2PteGlun complexes and does not result primarily from depletion of reduced folates because of dihydrofolate reductase inhibition. Because this complex has a slow rate of dissociation, dTMP synthetase activity would still be inhibited for some time after an influx of reduced folates (as in leucovorin rescue) sufficient to support the enzyme reaction under normal conditions.

Animals↗
PubMed1980

Hydrodynamic behavior of human and bacterial thymidylate synthetases and thymidylate synthetase--5-fluoro-2'-deoxyuridylate--5,10-methylenetetrahydrofolate complexes. Evidence for large conformational changes during catalysis.

The conformations of thymidylate synthetases from CCRF-CEM human leukemic cells and Lactobacillus casei were studied by hydrodynamic methods. Although the human enzyme has a molecular weight of 70 000--72 500, somewhat smaller than that of the L. casei enzyme, it has a larger Stokes radius and a lower sedimentation coefficient, indicating that the human enzyme is less spherical than the bacterial enzyme. Thymidylate synthetases from the human leukemic cells and the bacterial source both undergo substantial conformational changes upon the formation of a covalent ternary complex with the mechanism-based inhibitor 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate. The Stokes radius of both proteins decreases by 3.5% when the ternary complex is formed in spite of the 1.8% increase in molecular weight, and the sedimentation coefficient increases by 3.5% after appropriate corrections for the bound ligands. Ternary complex formation results in a more compact structure for both enzymes, with approximately the same reduction in the frictional ratio. Experiments with the bacterial enzyme indicate that approximately 70% of the total conformational change occurs upon binding of 1 mol of ligands/mol of enzyme. These results demonstrate that human and bacterial thymidylate synthetases undergo marked structural changes upon forming a ternary complex which is probably very similar to an activated complex formed with both substrates. These investigations also provide evidence for fundamental similarities in the mechanism of ternary complex formation with 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate, despite the marked differences in amino acid composition and the dissimilar conformations of these two enzymes obtained from widely divergent sources.

Binding Sites↗
PubMed1979

Thymidylate synthetase and 2'-deoxyuridylate form a tight complex in the presence of pteroyltriglutamate.

Thymidylate synthetases of human and bacterial origin form a tightly bound complex with the substrate dUMP in the presence of pteroyltriglutamate. This complex and the weaker enzyme . dUMP binary complex can be isolated and conveniently assayed by nitrocellulose disc filtration using [6-3H]dUMP as the radioactive ligand. Intact thymidylate synthetase . dUMP . pteroyltriglutamate complex can be obtained by gel filtration chromatography on Sephadex G-25, but the binary enzyme . dUMP complex dissociates under the same conditions. Scatchard plots show the presence of two nonequivalent dUMP binding sites on the enzyme for the pteroyltriglutamate complex, with dissociation constants of 5 and 95 nM compared to 730 nM for the binary complex. The implications of these findings for folate analog inhibition of thymidylate synthetase are discussed.

Cell Line↗
PubMed1979

Thymidylate synthetase purified to homogeneity from human leukemic cells.

Thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) from a human leukemic cell line has been purified to homogeneity with one-step affinity column chromatography. The purified enzyme has a specific activity of 3.8 micron/min per mg of protein, which corresponds to a turnover number of 250. These are the highest values reported for a thymidylate synthetase from neoplastic tissue. A ratio of 1.7 mol of 5-fluoro-2'-deoxyuridylate binds per mol of enzyme in the presence of 5,10-methylenetetrahydrofolate. The ternary complex so formed migrates intact on denaturing gels and can be precipitated with trichloroacetic acid; however, urea dissociates the ternary complex. The human thymidylate synthetase is composed of two subunits of 33,000 daltons each. It contains more residues of cysteine, glycine, and arginine and fewer of histidine than the well-studied thymidylate synthetase from Lactobacillus casei.

Cations↗
PubMedDate not supplied

Testing prospective anticancer drugs against human tumors using simple in vivo models.

The activities of established and experimental antitumor drugs were tested against the BRO and MeWo human melanomas transplanted in the subrenal capsule of phenotypically immunocompetent (CBA x C57BL/6)F1 mice. Immunosuppression was achieved with preliminary whole-body radiation, and cytological examination showed that the tumors of untreated mice consisted largely of viable tumor cells. Several drugs tested showed moderate activity against one or both tumors. The system described provides a basis for testing anticancer agents against a panel of human melanomas implanted in immunocompetent mice.

Animals↗
PubMedDate not supplied

Selective cytotoxicity of 5-hydroxyuridine for human colon adenocarcinoma cells.

The cytotoxicity of 5-hydroxyuridine (OHUrd), 5-FU, and 5-fluorodeoxyuridine was determined by colony assays for three human colon adenocarcinoma cell lines and for a cell line derived from normal fetal intestinal cells. All three tumor cell lines were more sensitive to OHUrd than were the FeInt cells, whereas 5-FU was more toxic to the latter. 5-Fluorodeoxyuridine was substantially more cytotoxic to only one of the tumor cell lines compared to the normal cells. In HT-29 tumor cells, OHUrd cytotoxicity could be prevented by coincubation with uridine or cytidine but not by pyrimidine deoxyribonucleosides or by purine nucleosides. Compared to OHUrd, 5-hydroxyuracil was much less cytotoxic for HT-29 cells. The increased cytotoxicity of OHUrd for these tumor cells compared to fetal intestinal cells may implicate biochemical differences that might be exploitable for improved chemotherapy. Direct comparison of drug sensitivity of colon tumor cells compared to normal counterpart cells may provide a method of screening agents selective for these tumor cells.

Adenocarcinoma↗