Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
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Affinity labeling of nucleotide-binding enzymes/proteins with 32P-labeled nucleotides is a powerful technique to identify nucleotide-binding proteins as well as to radiolabel the specific binding site. We have used this approach for labeling a nucleotide-binding domain in DNA polymerase and have isolated peptides bearing the linked nucleotides. The method used for separating tryptic peptides on hydrophobic matrices with an acetonitrile gradient in 0.1% trifluoroacetic acid as eluent results in loss of radioactivity, presumably through dissociation of the cross-linked nucleotide. This can be averted by the use of a non-acidic medium in the peptide purification protocol. We have devised a relatively simple procedure to concentrate the nucleotide-linked peptides by chromatography on DEAE-Sephadex A25. Most neutral and basic peptides as well as free nucleotides are removed by eluting the DEAE-Sephadex column with 0.2 M ammonium bicarbonate. The nucleotide-linked peptide is then eluted with 0.6 M ammonium bicarbonate. Radioactivity in the collected fractions is conveniently determined by scintillation counting. Labeled peptide in the 0.6 M ammonium bicarbonate eluate can be purified on a C4 reversed-phase column with an acetonitrile gradient in phosphate buffer (pH 6.8). By this procedure, 32P-labeled nucleotide linked with protein/peptide can be quantitatively purified with minimum loss.
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PMID:Purification of nucleotide-linked peptide. 306 Apr 74

1-O-Acetyl-2,5-di-O-p-chlorobenzoyl-3-deoxy-D-ribofuranose (1), derived from the antibiotic cordycepin was coupled with trimethylsilylated derivatives (2a-c) of N4-propionylcytosine, N4-p-toluoyl-5-fluorocytosine and 5-fluorouracil in the presence of trimethylsilyl trifluoromethanesulfonate (TMS-triflate) to give fully acylated nucleosides (3a-b and 3d, respectively). Selective removal of the N4-propionyl group of 3a by treatment with hydrazine hydrate gave 2',5'-di-O-p-chlorobenzoyl-3'-deoxycytidine (4). Deamination of 4 with sodium nitrite in trifluoroacetic acid afforded 2',5'-di-O-p-chlorobenzoyluridine (3c) in good yield. Compounds 3a-d were saponified to give free 3'-deoxycytidine (5a), 5-fluoro-3'-deoxycytidine (5b), 3'-deoxyuridine (5c), and 5-fluoro-3'-deoxyuridine (5d), respectively. These 3'-deoxyribonucleosides (5a-d) were then converted to corresponding 5'-monophosphate and further phosphorylated to the 5'-triphosphates by the phosphoroimidazolidate method. The nucleosides (5a-d) were examined for growth-inhibitory effects on mouse leukemic L5178Y cells, and their IC50 values (microgram/ml) were 1.8, 33, 6.5, and 18, respectively. On the other hand, the antiviral activities of these compounds on a rhabdovirus, infectious hematopoietic necrosis virus (IHNV), were moderate (IC50 = 100-500 micrograms/ml in CHSE-214 cells). The 5'-triphosphates showed remarkable inhibitory effects on DNA polymerase beta and DNA polymerase alpha-primase purified from testes of the cherry salmon, Oncorhynchus masou, but not on common DNA polymerase alpha from same source.
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PMID:Synthetic nucleosides and nucleotides. XXXV. Synthesis and biological evaluations of 5-fluoropyrimidine nucleosides and nucleotides of 3-deoxy-beta-D-ribofuranose and related compounds. 857 37