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61.
I B Bronshte?n A M Shuster L V Shevchenko I I Gromova O A Kvashuk O N Geva A G Gabinov 《Molekuliarnaia biologiia》1989,23(6):1553-1557
Immunoscreening of the human placenta cDNA-library in the expression vector lambda gt11 using non-isotope detection based on the avidin-biotin system allowed to identify a number of clones encoding human topoisomerase I. The fusion protein from an extract of Escherichia coli cells infected with the recombinant phage lambda gt11 interacts with the monoclonal antibody raised against topoisomerase I from calf thymus; the dissociation constant being 5.7.10(-8) M. The restricted DNA fragments coding for the topoisomerase polypeptide in the composition of the fusion protein were recloned, and expression in the pEX vector was obtained. The functional analysis of the expression products has enabled localization of the epitope of binding the monoclonal antibody. It was demonstrated that the identified fusion protein can be applied for diagnosis of autoimmune diseases. 相似文献
62.
Sequence dependent modulating effect of camptothecin on the DNA-cleaving activity of the calf thymus type I topoisomerase. 总被引:3,自引:2,他引:1
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I I Gromova V L Buchman R A Abagyan A V Ulyanov I B Bronstein 《Nucleic acids research》1990,18(3):637-645
High-resolution mapping of topol cleavages in the regions of human DNA including the oncogene c-Ha-ras and p53, has revealed three kinds of topol cleavage sites: cleavage sites not affected by camptothecin; cleavage sites reinforced only in the presence of camptothecin, and cleavage sites which weaken in the presence of camptothecin. Statistical analysis of sequences revealed certain nucleotide or dinucleotide preferences for three groups studied. The preferences in camptothecin-reduced sites predominate upstream from the cleavage point, whereas in camptothecin-induced sites the situation is reversed. The influence of camptothecin on cleavage sites induced by two molecular forms of topol has been also studied. 相似文献
63.
M N Vinogradova E S Gromova O I Griaznova M G Isaguliants S A Kuznetsova 《Bioorganicheskaia khimiia》1987,13(9):1194-1204
Ability of the EcoRII restriction endonuclease to cleave 14-base-pair DNA duplexes with nucleotide substitutions in the recognition site CCA/TGG and in the adjacent base pair has been studied. Modifications leading to a local change in the substrate conformation (rU residue in and outside the recognition site, A.A- or A.C-pairs in the flanking sequence) reduce the rate of hydrolysis, the effect being maximal when the modified base pair is outside the recognition site. No digestion occurs when the internal dC-residue of the recognition site is 5-methylated in one or both strands. Replacement of dT residue in the EcoRII recognition site by dfl5U residue results in a dramatic inhibition of hydrolysis. Km and kcat for the cleavage of 14-base-pair DNA duplex have been determined. The cleavage rate of the dT-containing strand of the recognition site in 1.5 fold higher comparing with the dA-containing strand. The cleavage of both strands of the substrate by EcoRII endonuclease is confirmed to proceed in one enzyme-substrate complex. 相似文献
64.
O V Petrauskene E A Kubareva E S Gromova Z A Shabarova 《European journal of biochemistry》1992,208(3):617-622
The efficiency of cleavage of DNA duplexes with single EcoRII recognition sites by the EcoRII restriction endonuclease decreases with increasing substrate length. DNA duplexes of more than 215 bp are not effectively cleaved by this enzyme. Acceleration of the hydrolysis of long single-site substrates by EcoRII is observed in the presence of 11-14-bp substrates. The stimulation of hydrolysis depends on the length and concentration of the second substrate. To study the mechanism of EcoRII endonuclease stimulation, DNA duplexes with base analogs and modified internucleotide phosphate groups in the EcoRII site have been investigated as activators. These modified duplexes are cleaved by EcoRII enzyme with different efficiencies or are not cleaved at all. It has been discovered that the resistance of some of them can be overcome by incubation with a susceptible canonical substrate. The acceleration of cleavage of long single-site substrates depends on the type of modification of the activator. The modified DNA duplexes can activate EcoRII catalyzed hydrolysis if they can be cleaved by EcoRII themselves or in the presence of the second canonical substrate. It has been demonstrated that EcoRII endonuclease interacts in a cooperative way with two recognition sites in DNA. The cleavage of one of the recognition sites depends on the cleavage of the other. We suggest that the activator is not an allosteric effector but acts as a second substrate. 相似文献
65.
Summary Two molecular forms of topoisomerase I differing in size and sensitivity to camptothecin were isolated from calf thymus. Mapping of topo I cleavage sites of the cloned chicken A-globin and human c-Ha-ras genes was carried out. Camptothecin was shown to affect site specificity of the topoisomerases. 相似文献
66.
Aminasine, BeSO4 and Pt-5-sulfomercaptoquinolinate action on Ca-ATPase of SR showed a considerably less inhibiting effect as compared with that produced on the native membranes. The inhibiting action of the chemical compounds on those of native SR membranes is followed by the increase of mobility of hydrophobic segments of the membrane. The kinetic study of ATPase reaction at various temperatures showed on low-temperature transformation after the action by chemical compounds. Both structural transformations retain in the modified SR membrane independent of the chemical treatment. The activation energies considerably differ from those of native an modified membranes without chemical treatment (particularly in the region of 10-20 degrees). The data obtained allow to suggest that the inhibiting action of chemical compounds is followed by the changes in microviscosity (in the region of protein-lipid interaction of SR membrane, in particular), which by conformation transformations affect the configuration of the enzyme active center, alternating its geometry and catalytic activity. 相似文献
67.
Jane L Wagstaff Jonathan N Pruneda Stefan MV Freund David Komander 《The EMBO journal》2017,36(24):3555-3572
The Ser/Thr protein kinase PINK1 phosphorylates the well‐folded, globular protein ubiquitin (Ub) at a relatively protected site, Ser65. We previously showed that Ser65 phosphorylation results in a conformational change in which Ub adopts a dynamic equilibrium between the known, common Ub conformation and a distinct, second conformation wherein the last β‐strand is retracted to extend the Ser65 loop and shorten the C‐terminal tail. We show using chemical exchange saturation transfer (CEST) nuclear magnetic resonance experiments that a similar, C‐terminally retracted (Ub‐CR) conformation also exists at low population in wild‐type Ub. Point mutations in the moving β5 and neighbouring β‐strands shift the Ub/Ub‐CR equilibrium. This enabled functional studies of the two states, and we show that while the Ub‐CR conformation is defective for conjugation, it demonstrates improved binding to PINK1 through its extended Ser65 loop, and is a superior PINK1 substrate. Together our data suggest that PINK1 utilises a lowly populated yet more suitable Ub‐CR conformation of Ub for efficient phosphorylation. Our findings could be relevant for many kinases that phosphorylate residues in folded protein domains. 相似文献
68.
O. M. Gritsenko S. N. Mikhailov E. V. Efimtseva A. Van Aerschot P. Herdewijn E. S. Gromova 《Nucleosides, nucleotides & nucleic acids》2013,32(10-12):1805-1820
Abstract Affinity labeling of methyltransferase MvaI by DNA duplexes containing oxidized 2′-O-β-D-ribofuranosylcytidine or 1-(β-D-galactopyranosyl)thymine residues was performed. Partial chemical hydrolysis of the covalently bound methylase in the conjugates with the dialdehyde-containing DNA allowed us to determine the amino acid region in the C terminus of methylase MvaI that interacts with DNA. 相似文献
69.
Recovery cycles of primary evoked potentials to light flashes in the visual cortical area of waking rats were studied under conditions of pharmacological and electrical influences on serotonin (5-HT)- and noradren (NA)ergic brain systems. All factors used induced oscillations of the recovery cycles. Periods of oscillations were similar (300-400 ms) during pharmacological suppression of the NA-system and during high-frequency (500 Hz) electrical stimulation or lesion of locus coeruleus. Analogous influences on 5-HT-system were accompanied by oscillations of recovery cycles with a period of 200 ms. Mechanism of inhibitory action of high-frequency electrical stimulation on activity of monoaminergic systems is discussed. 相似文献
70.
E S Gromova E A Kubareva A A Elov E A Akatova I I Nikol'skaia Z A Shabarova 《Biokhimii?a (Moscow, Russia)》1991,56(3):552-559
The interaction of enzymes SsoII (decreases CCNGG) and MvaI (CC decreases A/TGG) with concatemeric DNA duplexes used earlier to study EcoRII (decreases CCA/TGG) TGG was investigated with a view of elucidating the general principles of the restriction endonuclease function. A pattern common for all the three enzymes was observed with DNA duplexes containing AA or TT pairs in the central position of the recognition site. The AA pair blocks or substantially hinders the endonuclease action, whereas the TT pair is either less inhibitory or altogether inert. SsoII, similar to EcoRII was able to processively cleave the concatemeric substrates and to interact with (or to be close to) the hydrogen in the 5th position of the outer dC residue of the recognition site. MvaI was found to differ from EcoRII in the way they recognize and cleave the same nucleotide sequence. The substrate-bound MvaI molecule is incapable of linear diffusion along the DNA. Effective hydrolysis of dU- and m5dC-containing polymers rules out the participation of hydrophobic contacts of the enzyme with the methyl group of the dT residue and with the 5th hydrogen of the outer dC residue of the recognition site in DNA-protein interactions. 相似文献