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Panova N. G. Shcheveleva E. V. Alexeev C. S. Mukhortov V. G. Zuev A. N. Mikhailov S. N. Esipov R. S. Chuvikovsky D. V. Miroshnikov A. I. 《Molecular Biology》2004,38(5):770-776
The new substrates 4-thiouridine and 4-thiothymidine were proposed for spectrophotometric measurement of the activity of uridine (UP) and thymidine (TP) phosphorylases. At pH 7.5, 4-thiouridine has an absorbance maximum at 330 nm, and the difference in extinction coefficient () between 4-thiouridine and 4-thiouracil is 3000 –1cm–1. 4-Thiouridine proved to be a good substrate for UP: the Michaelis (
) and catalytic (k
cat) constants were estimated respectively at 130 M and 49 s–1 at 25°C. Even a greater (5000 M–1cm–1 at 336 nm) was observed for the 4-thiothymidine/4-thiothymine pair. 相似文献
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Chuvikovsky DV Esipov RS Skoblov YS Chupova LA Muravyova TI Miroshnikov AI Lapinjoki S Mikhailopulo IA 《Bioorganic & medicinal chemistry》2006,14(18):6327-6332
Ribokinase (RK) was expressed in the Escherichia coli ER2566 cells harboring the constructed expression plasmid encompassing the rbsK gene, encoding ribokinase. The recombinant enzyme was purified from sonicated cells by double chromatography to afford a preparation that was ca. 90% pure and had specific activity of 75 micromol/min mg protein. Catalytic activity of RK: (i) is strongly dependent on the presence of monovalent cations (potassium>ammonium>cesium), and (ii) is cooperatively enhanced by divalent magnesium and manganese ions. Besides D-ribose and 2-deoxy-D-ribose, RK was found to catalyze the 5-O-phosphorylation of D-arabinose, D-xylose, and D-fructose in the presence of ATP, and potassium and magnesium ions; L-ribose and L-arabinose are not substrates for the recombinant enzyme. A new radiochemical method for monitoring the formation of D-pentofuranose-5-[32P]phosphates in the presence of [gamma-32P]ATP and RK is reported. 相似文献
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Roman S Esipov Alexandr I Gurevich Dmitry V Chuvikovsky Larisa A Chupova Tatyana I Muravyova Anatoly I Miroshnikov 《Protein expression and purification》2002,24(1):56-60
The Escherichia coli genes encoding purine nucleoside phosphorylase, uridine phosphorylase, and thymidine phosphorylase were cloned into pET plasmids to generate highly effective E. coli BL21(DE3) strains producing each of these enzymes. Optimum conditions for biosynthesis of each enzyme as a soluble protein with intact biological activity were found. The crude preparations are approximately 80% pure and can be used immediately for enzymatic transglycosylation. The enzyme preparations were purified to homogeneity by two steps including fractional precipitation with ammonium sulfate and subsequent chromatography on Sephadex G-100 and DEAE-Sephacel. 相似文献
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Esipov RS Chupova LA Shvets SV Chuvikovsky DV Gurevich AI Muravyova TI Miroshnikov AI 《Protein and peptide letters》2003,10(4):404-411
The plasmid DNA pERilox4 containing the gene of the recombinant protein, which included the leader sequence and the oxytocinoyl lysine tetramer, was constructed. The high level of gene expression in E. coli was achieved. The method for purification of the recombinant protein and its isolation in the soluble form was developed. The conditions for digestion of the hybrid protein by trypsin and carboxypeptidase B were matched. The effective method for transformation of oxytocinic acid to oxytocin was worked out. The scheme suggested allowed obtaining oxytocin in high yield. 相似文献
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Esipov R. S. Gurevich A. I. Stepanenko V. N. Chupova L. A. Chuvikovsky D. V. Miroshnikov A. I. 《Russian Journal of Bioorganic Chemistry》2004,30(5):431-435
An artificial gene encoding thymosin 1 was obtained by chemoenzymatic synthesis and cloned into Escherichia coli. An expressing recombinant plasmid containing the hybrid protein gene, which encodes amino acid sequences of thymosin 1 and the Saccharomyces
cerevisiae intein Sce VMA, was constructed. The expression of the hybrid protein from the resulting hybrid gene in E. coli, the properties of the resulting hybrid protein, and the conditions for its nonenzymatic cleavage to thymosin 1 were studied. 相似文献
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