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V G Debabov

Publications and source records attributed to V G Debabov.

At least 37 records · Page 2Linked to original sources
PubMed1994

Enzyme-catalyzed uridine phosphorolysis: SN2 mechanism with phosphate activation by desolvation.

The rate of uridine phosphorolysis catalyzed by uridine phosphorylase from Escherichia coli decreases with increasing ionic strength. In contrast, the rate was increased about twofold after preincubation of uridine phosphorylase with 60% acetonitrile. These data correlate with known effects of polar and bipolar aprotic solvents on SN2 nucleophilic substitution reactions. The enzyme modified with fluorescein-5'-isothiocyanate (fluorescein residue occupies an uridine-binding subsite [Komissarov et al., (1994) Biochim. Biophys. Acta 1205, 54-58]) was selectively modified with irreversible inhibitor SA-423, which reacts near the phosphate-binding subsite. The double-modified uridine phosphorylase is assumed to imitate the enzyme-substrate complex. Modification with SA-423 was accompanied with dramatic changes in the absorption spectrum of active site-linked fluorescein, which were identical to those for fluorescein in a hydrophobic medium, namely 80% acetonitrile. The data obtained suggest that an increase in active site hydrophobicity leads to phosphate desolvation and facilitates the enzymatic SN2 uridine phosphorolysis reaction.

Binding Sites↗
PubMed1994

Selective modification of putative uridine-binding site of uridine phosphorylase from E. coli with fluorescein 5'-isothiocyanate.

A putative uridine-binding site of uridine phosphorylase (EC 2.4.2.3) from E. coli was modified with fluorescein 5'-isothiocyanate (FITC). Treatment with FITC irreversibly inactivates the enzyme (Ki = 1.0 mM, k2 = 0.15 min-1). Under the conditions of 90% inactivation the incorporation of the reagent reaches about 1 mol per mol of the enzyme subunit. Addition of uridine prevents the enzyme inactivation by FITC. In contrast to this, addition of a second substrate phosphate increases the rate of inactivation by 2.3-fold (k2 = 0.34 min-1), but has no effect on the affinity of the reagent to the enzyme. The modified protein retains the ability to bind phosphate but not uridine. According to differential absorption spectroscopy data, the binding of phosphate to the active site of the enzyme is accompanied by conformational changes which may accelerate the inactivation rate. The data presented suggest that in the UPase FITC occupies the putative uridine-binding site, while the phosphate-binding site still retains the ability to interact with the second substrate.

Amino Acid Sequence↗
PubMed1994

Construction and expression in Escherichia coli of hybrid genes composed of sequences encoding diphtheria toxin and human CD4 receptor.

Derivatives of natural toxins possessing substituted receptor-recognition domains of different specificities can be used as instruments for the selective elimination of target cells. We have constructed two different types of hybrid genes that encode proteins composed of diphtheria toxin (DT) lacking the C-terminal residues that mediate toxin binding fused with the N-terminal region of human CD4 (Lys10 to Glu152). One of these hybrids encodes a protein with CD4 at the N terminus, while the other encodes a protein with CD4 at the C terminus. The stability of these two proteins was dramatically different. We could not detect a full-size product when the first construct was expressed in Escherichia coli. In contrast, proteins encoded by the second construct were more stable. In the latter case, the amount of full-size hybrid protein was 1-2% of the total cell protein. We speculate on the involvement of the region that resembles the processing site of Pseudomonas aeruginosa exotoxin A in the proteolytic degradation of the product encoded by the first type of hybrid.

Amino Acid Sequence↗
PubMed1991

[Site-directed mutagenesis with the use of addressed fragmentation of single-stranded DNA for obtaining hybrids of homologous proteins].

Site-directed multiple mutagenesis to obtain hybrids of homologous proteins was carried out by means of the oligonucleotide-targeting digestion of ss DNA. The procedure is more convenient, rapid and simple than the step-by-step approach. To demonstrate different approaches to targeting digestion of ss DNA, NcoI, HinfI, FokI endonucleases were used for DNA cleavage. A hydride of the human and porcine IFN-alpha-2-genes has been constructed.

Animals↗
PubMed1991

[Use of uracil-repair selection for extensive DNA sequence deletions].

A procedure for extensive deletion mutagenesis of DNA using the uracil repair system is exemplified by reconstruction of the pBR322 replication regulatory region cloned into M13tg131. By means of an oligonucleotide primer the 116-nucleotide fragment was excised and four nucleotides were introduced to form a BglII restriction site. Use of the uracil repair selection provided a 30-fold increase in the deletion mutagenesis efficiency.

Base Sequence↗
PubMed1990

[Development of the vector-host system in Corynebacterium. Cloning and expression of homoserine dehydrogenase and homoserine kinase genes in Corynebacterium cells].

Novel cloning vectors for glutamic acid producing bacteria have been constructed. The cryptic plasmid pBO1 (4.4 kb) from Brevibacterium sp. recombined with the plasmid pACYC184 (4.0 kb) from Escherichia coli was used to produce composite plasmid named pKA1. The plasmid could propagate and express the Cm-r phenotype in E. coli and coryneform glutamic acid producing bacteria Br. flavum, C. glutamicum, Br. lactofermentum. The pKA1 plasmid and its variants deleted within non-essential plasmid regions with unique restriction sites HindIII, SalGI, SphI were used in cloning experiments. The genes coding for threonine biosynthesis of C. glutamicum and Br. flavum were subcloned into shuttle vectors in C. glutamicum cells. Recombinant plasmids were introduced into protoplasts by polyethylenglycol-mediated transformation of plasmid DNAs. It was shown that the presence of plasmids containing the Br. flavum thrA2 gene in C. glutamicum (thrB) caused 10-fold increase in homoserine dehydrogenase activity, as compared to that of wild type strain, and in homoserine production.

Alcohol Oxidoreductases↗
PubMed1989

Phasmids as effective and simple tools for construction and analysis of gene libraries.

Phasmid lambda pMYF131, a hybrid of phage lambda vectors and plasmid pUC19, was constructed. The phasmid and its derivatives were shown to be efficient vectors for construction and analysis of gene libraries in Escherichia coli cells. The lambda pMYF131 DNA molecule contains all the genes and regions essential for phage lytic development. The plasmid cannot be packaged either in the monomeric or the oligomeric form due to its specific length. Elongation of the DNA molecule by ligation with fragments of foreign DNA can make it packageable and this is easily detected by plaque formation. Hence, the procedures used to construct genomic libraries can be simplified by selection of only recombinant DNA molecules just at the time and on the basis of their packaging in vitro. The output of recombinant clones per vector molecule was several times higher for vector lambda pMYF131, compared to phage vector lambda L47.1AB, and attained 3 x 10(6) clones per micrograms DNA. Vector and recombinant phasmids can be obtained in large quantities in plasmid form. lambda pMYF131 contains nine unique restriction sites which allow the cloning of DNA fragments with blunt ends and of fragments with various types of cohesive ends, obtained by digestion with 14 prototype restriction enzymes. The maximal size of the cloned DNA fragments is approx. 20 kb for lambda pMYF131. Phasmid vectors were used to construct libraries of bovine, pig and quail genomes, and genomic libraries of 17 species of bacteria. Application of suitable methods allowed the identification 13 individual genes within these libraries.

Bacteriophage lambda↗
PubMed1989

Cloning and analysis of structural and regulatory pectate lyase genes of Erwinia chrysanthemi ENA49.

Erwinia chrysanthemi ENA49 structural and regulatory ptl genes, coding for pectate lyase (Ptl) were cloned in Escherichia coli cells. Phage vector lambda L47.1 and phasmid vector lambda pMYF131 were used for constructing libraries of BamHI and EcoRI fragments, respectively, of Er. chrysanthemi chromosomal DNA. Among the 1,100 hybrid clones containing BamHI Er. chrysanthemi DNA fragments and 11,000 hybrid clones containing EcoRI fragments, six and 45 clones, respectively, were identified as having pectolytic activity. Two different structural genes, designated ptlA and ptlB, have been subcloned on multi-copy plasmids. Genes ptlA and ptlB are located side by side on the chromosome of Er. chrysanthemi and transcribe in the same direction. Each of the genes has its own promoter. Southern-blot hybridization analysis showed that the cloned ptl genes shared practically no homology and each of the genes was represented by a single copy on the Er. chrysanthemi chromosome. Other ptl genes capable of expression in E. coli cells were not found in the gene libraries. Negative regulation of the ptlA gene expression by a cloned gene called ptlR was shown. To screen the gene library for the ptlR gene, a specific genetic system was devised. The genes studied are located within an EcoRI chromosomal DNA fragment of 7.3 kb in the order: ptlA-ptlB-ptlR.

Bacteriophage lambda↗