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Vern L. Schramm - One of the best experts on this subject based on the ideXlab platform.
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Transition State Analysis of Thymidine Hydrolysis by Human Thymidine Phosphorylase
Journal of the American Chemical Society, 2010Co-Authors: Phillip A. Schwartz, Mathew J. Vetticatt, Vern L. SchrammAbstract:Human Thymidine phosphorylase (hTP) is responsible for Thymidine (dT) homeostasis, and its action promotes angiogenesis. In the absence of phosphate, hTP catalyzes a slow hydrolytic depyrimidination of dT yielding thymine and 2-deoxyribose (dRib). Its transition state was characterized using multiple kinetic isotope effect (KIE) measurements. Isotopically enriched Thymidines were synthesized enzymatically from glucose or (deoxy)ribose, and intrinsic KIEs were used to interpret the transition state structure. KIEs from [1′-14C]-, [1-15N]-, [1′-3H]-, [2′R-3H]-, [2′S-3H]-, [4′-3H]-, and [5′-3H]dTs provided values of 1.033 ± 0.002, 1.004 ± 0.002, 1.325 ± 0.003, 1.101 ± 0.004, 1.087 ± 0.005, 1.040 ± 0.003, and 1.033 ± 0.003, respectively. Transition state analysis revealed a stepwise mechanism with a 2-deoxyribocation formed early and a higher energetic barrier for nucleophilic attack of a water molecule on the high energy intermediate. An equilibrium exists between the deoxyribocation and reactants prior to t...
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nucleophilic participation in the transition state for human Thymidine phosphorylase
Journal of the American Chemical Society, 2004Co-Authors: Matthew R. Birck, Vern L. SchrammAbstract:Recombinant human Thymidine phosphorylase catalyzes the reaction of arsenate with Thymidine to form thymine and 2-deoxyribose 1-arsenate, which rapidly decomposes to 2-deoxyribose and inorganic arsenate. The transition-state structure of this reaction was determined using kinetic isotope effect analysis followed by computer modeling. Experimental kinetic isotope effects were determined at physiological pH and 37 °C. The extent of forward commitment to catalysis was determined by pulse-chase experiments to be 0.70%. The intrinsic kinetic isotope effects for [1‘-3H]-, [2‘R-3H]-, [2‘S-3H]-, [4‘-3H]-, [5‘-3H]-, [1‘-14C]-, and [1-15N]-Thymidines were determined to be 0.989 ± 0.002, 0.974 ± 0.002, 1.036 ± 0.002, 1.020 ± 0.003, 1.061 ± 0.003, 1.139 ± 0.005, and 1.022 ± 0.005, respectively. A computer-generated model, based on density functional electronic structure calculations, was fit to the experimental isotope effect. The structure of the transition state confirms that human Thymidine phosphorylase proceeds ...
Jon Nissen-meyer - One of the best experts on this subject based on the ideXlab platform.
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Thymidine secretion by hybridoma and myeloma cells
Biochemical and Biophysical Research Communications, 2006Co-Authors: Bjørn Spilsberg, Frode Rise, Dirk Petersen, Jon Nissen-meyerAbstract:Abstract Secretion of Thymidine appeared to be a common property of hybridoma and myeloma cells, but not of other cell types, which were tested. Of three hybridoma cell lines tested, all secreted Thymidine in amounts resulting in the accumulation of Thymidine to concentrations of 10–20 μM in the culture medium. Also three of five myeloma cell lines that were analyzed secrete Thymidine, but none of the other cell types that were studied. Thymidine was purified to homogeneity (4 mg purified from 3 l of culture medium) and identified as such by nuclear magnetic resonance spectroscopy. The cells that secreted Thymidine showed high resistance to the growth inhibitory effect of Thymidine.
H. Reuveni - One of the best experts on this subject based on the ideXlab platform.
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Thymidine salvage changes with differentiation in human keratinocytes in vitro.
The Journal of investigative dermatology, 1991Co-Authors: Pauline M. Schwartz, Steven K. Barnett, H. ReuveniAbstract:Abstract We compared the capacity of proliferating and differentiating keratinocytes to salvage and catabolize extracellular Thymidine. Both populations of cells catabolized Thymidine to thymine and possessed Thymidine phosphorylase activity. As keratinocytes differentiate, Thymidine phosphorylase activity ultimately increased twofold. In contrast, proliferating and differentiating keratinocytes differed markedly in their capacity to salvage extracellular Thymidine. Proliferating keratinocytes readily salvaged extracellular Thymidine to form nucleotides, whereas differentiating cells rapidly lost this capacity. The inability of differentiating cells to form nucleotides from Thymidine was not attributed to reduced availability of Thymidine due to catabolism but rather was the result of the rapid loss of Thymidine kinase activity. As keratinocytes differentiate in suspension culture, they lose 41% of Thymidine kinase activity in 8 h and over 90% of activity in 12 h. Our data indicate that loss of capacity to salvage extracellular Thymidine for synthesis of nucleotides closely parallels the onset of differentiation in keratinocytes.
Bjørn Spilsberg - One of the best experts on this subject based on the ideXlab platform.
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Thymidine secretion by hybridoma and myeloma cells
Biochemical and Biophysical Research Communications, 2006Co-Authors: Bjørn Spilsberg, Frode Rise, Dirk Petersen, Jon Nissen-meyerAbstract:Abstract Secretion of Thymidine appeared to be a common property of hybridoma and myeloma cells, but not of other cell types, which were tested. Of three hybridoma cell lines tested, all secreted Thymidine in amounts resulting in the accumulation of Thymidine to concentrations of 10–20 μM in the culture medium. Also three of five myeloma cell lines that were analyzed secrete Thymidine, but none of the other cell types that were studied. Thymidine was purified to homogeneity (4 mg purified from 3 l of culture medium) and identified as such by nuclear magnetic resonance spectroscopy. The cells that secreted Thymidine showed high resistance to the growth inhibitory effect of Thymidine.
Phillip A. Schwartz - One of the best experts on this subject based on the ideXlab platform.
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Transition State Analysis of Thymidine Hydrolysis by Human Thymidine Phosphorylase
Journal of the American Chemical Society, 2010Co-Authors: Phillip A. Schwartz, Mathew J. Vetticatt, Vern L. SchrammAbstract:Human Thymidine phosphorylase (hTP) is responsible for Thymidine (dT) homeostasis, and its action promotes angiogenesis. In the absence of phosphate, hTP catalyzes a slow hydrolytic depyrimidination of dT yielding thymine and 2-deoxyribose (dRib). Its transition state was characterized using multiple kinetic isotope effect (KIE) measurements. Isotopically enriched Thymidines were synthesized enzymatically from glucose or (deoxy)ribose, and intrinsic KIEs were used to interpret the transition state structure. KIEs from [1′-14C]-, [1-15N]-, [1′-3H]-, [2′R-3H]-, [2′S-3H]-, [4′-3H]-, and [5′-3H]dTs provided values of 1.033 ± 0.002, 1.004 ± 0.002, 1.325 ± 0.003, 1.101 ± 0.004, 1.087 ± 0.005, 1.040 ± 0.003, and 1.033 ± 0.003, respectively. Transition state analysis revealed a stepwise mechanism with a 2-deoxyribocation formed early and a higher energetic barrier for nucleophilic attack of a water molecule on the high energy intermediate. An equilibrium exists between the deoxyribocation and reactants prior to t...
