Search PubMed⌕ Search

Biomedical subjects

L Kisselev

Publications and source records attributed to L Kisselev.

33 records · Page 2Linked to original sources
PubMed1996

Interferons induce accumulation of diadenosine triphosphate (Ap3A) in human cultured cells.

After incubation of human monocytes J96 and human myeloid leukemia HL60 cells with interferons (IFN) alpha or gamma, the Ap3A concentration considerably increases in parallel with accumulation of tryptophanyl-tRNA synthetase (TrpRS, EC 6.1.1.2). The Ap3A formation in response to IFNs is catalysed by an excessive amount of TrpRS. Although the Ap3A function still remains unknown, its accumulation may imply the Ap3A involvement in the IFN-signalling pathway.

Cell Line↗
PubMed1996

Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase.

Termination of translation in eukaryotes is governed by two polypeptide chain release factors, eRF1 and eRF3 on the ribosome. eRF1 promotes stop-codon-dependent hydrolysis of peptidyl-tRNA, and eRF3 interacts with eRF1 and stimulates eRF1 activity in the presence of GTP. Here, we have demonstrated that eRF3 is a GTP-binding protein endowed with a negligible, if any, intrinsic GTPase activity that is profoundly stimulated by the joint action of eRF1 and the ribosome. Separately, neither eRF1 nor the ribosome display this effect. Thus, eRF3 functions as a GTPase in the quaternary complex with ribosome, eRF1, and GTP. From the in vitro uncoupling of the peptidyl-tRNA and GTP hydrolyses achieved in this work, we conclude that in ribosomes both hydrolytic reactions are mediated by the formation of the ternary eRF1-eRF3-GTP complex. eRF1 and the ribosome form a composite GTPase-activating protein (GAP) as described for other G proteins. A dual role for the revealed GTPase complex is proposed: in " GTP state," it controls the positioning of eRF1 toward stop codon and peptidyl-tRNA, whereas in "GDP state," it promotes release of eRFs from the ribosome. The initiation, elongation, and termination steps of protein synthesis seem to be similar with respect to GTPase cycles.

Base Sequence↗
PubMed1995

Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3.

Termination of translation in higher organisms is a GTP-dependent process. However, in the structure of the single polypeptide chain release factor known so far (eRF1) there are no GTP binding motifs. Moreover, in prokaryotes, a GTP binding protein, RF3, stimulates translation termination. From these observations we proposed that a second eRF should exist, conferring GTP dependence for translation termination. Here, we have shown that the newly sequenced GTP binding Sup35-like protein from Xenopus laevis, termed eRF3, exhibits in vitro three important functional properties: (i) although being inactive as an eRF on its own, it greatly stimulates eRF1 activity in the presence of GTP and low concentrations of stop codons, resembling the properties of prokaryotic RF3; (ii) it binds and probably hydrolyses GTP; and (iii) it binds to eRF1. The structure of the C-domain of the X.laevis eRF3 protein is highly conserved with other Sup35-like proteins, as was also shown earlier for the eRF1 protein family. From these and our previous data, we propose that yeast Sup45 and Sup35 proteins belonging to eRF1 and eRF3 protein families respectively are also yeast termination factors. The absence of structural resemblance of eRF1 and eRF3 to prokaryotic RF1/2 and RF3 respectively, may point to the different evolutionary origin of the translation termination machinery in eukaryotes and prokaryotes. It is proposed that a quaternary complex composed of eRF1, eRF3, GTP and a stop codon of the mRNA is involved in termination of polypeptide synthesis in ribosomes.

Amino Acid Sequence↗
PubMed1995

Localization of human ARF2 and NCK genes and 13 other NotI-linking clones to chromosome 3 by fluorescence in situ hybridization.

Two human genes containing NotI sites, ADP-ribosylation factor (ARF2) and melanoma NCK protein, were mapped by fluorescence in situ hybridization to 3p21.2-->p21.1 and 3q21, respectively. Thirteen other NotI-linking clones, representing sequence tagged sites, were also mapped to different regions of human chromosome 3. Two of these clones that contain sequences 80% homologous to the rat tropoelastin gene and brain Cl- channel protein CLC-2 gene probably represent new human genes closely related to the known rat genes.

ADP-Ribosylation Factors↗
PubMed1994

P1,P3-bis(5'-adenosyl)triphosphate (Ap3A) as a substrate and a product of mammalian tryptophanyl-tRNA synthetase.

Bovine tryptophanyl-tRNA synthetase (TrpRS, E.C.6.1.1.2) is unable to catalyze in vitro formation of Ap4A in contrast to some other aminoacyl-tRNA synthetases. However, in the presence of L-tryptophan, ATP-Mg2+ and ADP the enzyme catalyzes the Ap3A synthesis via adenylate intermediate. Ap3A (not Ap4A) may serve as a substrate for TrpRS in the reaction of E.(Trp approximately AMP) formation and in the tRNA(Trp) charging. The Km value for Ap3A was higher than the Km for ATP (approx. 1.00 vs. 0.22 mM) and Vmax was 3 times lower than for ATP. The Zn(2+)-deficient enzyme catalyzes Ap3A synthesis in the absence of exogenous ADP due to ATPase activity of Zn(2+)-deprived TrpRS. The inability of mammalian TrpRS to synthesize Ap4A, might be considered as a molecular tool preventing the removal of Zn2+ due to chelation by Ap4A and therefore preserving the enzyme activity.

Adenosine Triphosphate↗
PubMed1993

Nucleoside triphosphatase activity associated with the N-terminal domain of mammalian tryptophanyl-tRNA synthetase.

Bovine tryptophanyl-tRNA synthetase (EC 6.1.1.2) deprived of Zn2+ by chelation with the phosphonate analog of Ap4A hydrolyzed ATP(GTP) to ADP(GDP) although its ability to form tryptophanyl adenylate was impaired. This hydrolytic activity is stimulated by Mg2+ and Mn2+ ions and inhibited by Zn2+. Monoclonal antibody Am1 against the N-terminal domain of the enzyme completely abolished ATP(GTP)ase activity. The core peptide generated after proteolytic splitting of the N-domain lacks this activity. We suggest that the nucleotide binding site(s) different from ATP sites involved in aminoacylation reaction reside(s) at the N-terminal domain(s) of the enzyme.

Adenosine Triphosphatases↗
PubMed1993

Random-splitting of tRNA transcripts as an approach for studying tRNA-protein interactions.

Location of phosphodiester bonds essential for aminoacylation of bovine tRNA(Trp) was identified using a randomly cleaved transcript synthesized in vitro. It was found that cleavage of phosphodiester bonds after nucleotides in positions 21, 22, 36-38, 57-59, 62 and 64 were critical for aminoacylation capacity of tRNA(Trp)-transcript. These cleavage sites were located in the regions of tRNA molecule protected by the cognate synthetase against chemical modification and in the regions presumably outside the contact area as well. These results indicate that for maintenance of aminoacylation ability the intactness of the certain regions of the tRNA backbone structure is necessary. Random splitting of non-modified RNA with alkali followed by separation of active and inactive molecules and identification of cleavage sites developed in this work may become a general approach for studying the role of RNA covalent structure in its interaction with proteins.

Acylation↗
PubMed1993

Interferon inducibility of mammalian tryptophanyl-tRNA synthetase: new perspectives.

Mammalian aminoacyl-tRNA synthetases are indispensible components of the cell's protein-synthesizing machinery. Surprisingly, recent experiments have demonstrated that synthesis of tryptophanyl-tRNA synthetase (WRS) is markedly enhanced after incubation of human cells with interferons. Why is this housekeeping enzyme interferon-inducible? Several hypotheses have been suggested. One hypothesis, that premature termination of protein synthesis was involved, was boosted by the discovery that the deduced amino acid sequence of the mammalian peptide chain release factor (RF) closely resembled that of WRS. Further investigation, however, suggests that the DNA encoding RF was wrongly identified and in fact encodes a rabbit WRS subunit. Other hypotheses on the interferon-inducibility of WRS, including the possibility that the protein performs other, regulatory functions in addition to its core enzymic activity, remain to be explored.

Amino Acid Sequence↗
PubMed1987

Monoclonal antibodies against RNA-dependent DNA polymerase from myeloblastosis-associated viruses.

Two lines of hybridomas, RT-12 and RT-14, secreting monoclonal antibodies against the reverse transcriptase from myeloblastosis-associated viruses have been prepared. The monoclonal antibodies RT-12 and RT-14 specifically react with reverse transcriptase, as has been shown by radioimmunoassay and enzyme-linked immunoassay techniques after SDS-PAGE and blotting to nitrocellulose membranes. It has been shown that antigenic determinants for RT-12 and RT-14 are stable to SDS denaturation, hence they belong to a linear type; they are located on the alpha subunit of the enzyme.

Animals↗
PubMed1983

Mutual conformational changes of tryptophanyl-tRNA synthetase and tRNATrp in the course of their specific interaction.

tRNATrp (beef, yeast) is capable of accelerating limited tryptic hydrolysis of the N-terminal part in the polypeptide chains of dimeric beef pancreas tryptophanyl-tRNA synthetase; it can also eliminate the protective effect of tryptophanyl adenylate on the enzyme proteolysis. The effect of tRNA on the proteolysis is manifested even when the 3'-CCA terminus is removed. It has been concluded that the conformation of the synthetase changes when it forms a complex with tRNATrp. Yeast tRNATrp lacking the 3'-half of the acceptor stem can still interact with the synthetase and, to certain extent, induces changes in the conformation of the latter. The susceptibility of single-stranded and double-stranded regions of tRNATrp to cleavage with endonucleases has been studied, and the results are indicative of the fact that, regardless of considerable differences in the nucleotide sequence of yeast and beef tRNATrp, their three-dimensional structures are similar. This fact is consistent with the finding that parameters for the interaction of these tRNAsTrp with beef tryptophanyl-tRNA synthetase are rather close. The three-dimensional structure of tRNATrp is altered when the enzyme forms a complex with it, as seen from (a) a change in the circular dichroic spectrum and (b) an elevated susceptibility of the anticodon and, apparently, acceptor stems to cleavage with nuclease. The conversion of exposed cytidine residues in tRNATrp into uridine residues results in a loss of the acceptor activity; the capability to accelerate limited tryptic hydrolysis of tryptophanyl-tRNA synthetase is also lost although the enzyme-substrate complex, as seen from circular dichroic spectra, can still be formed. The conversion of cytosine in the anticodon stem into uracil modifies the conformation of the anticodon stem. The anticodon arm (including the anticodon) and the acceptor stem play an essential role in the interaction between tRNATrp and tryptophanyl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗
PubMed1978

Enzymatic synthesis of DNA complementary to mitochondrial mRNA via reverse transcription.

The poly(A)-containing mitochondrial mRNAs of rat liver were tested for their ability to serve as templates for the DNA synthesis by means of reverse transcription in the presence of the oligo(dT) primer and the RNA-directed DNA-polymerase from avian myeloblastosis virus. The mT-mRNA does not support the DNA synthesis in the standard conditions sufficient for effective reverse transcription of rabbit globin mRNA and of poly(A) in the presence of oligo(dT) primers. After a mild alkaline treatment of the mRNA and subsequent polyadenylation of the 3'-termini of the generated fragments with ATP:RNA adenyltransferase from E.coli the poly(A) (+) polyribonucleotides are able to serve as templates for reverse transcription in the presence of oligo(dT) and the reverse transcriptase. A conclusion is made that a "structural stop" exists in mitochondrial mRNA non-translable regions adjacent to the poly(A) terminal sequence. The "structural stop" is suggested to be caused by post-transcriptional modification of mRNA (methylation, etc.) or by a particularly stable secondary structure in this region of the mRNA molecules.

Animals↗