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L P Gavrilova

Publications and source records attributed to L P Gavrilova.

At least 19 recordsLinked to original sources
PubMed1993

Study of localization of the protein-synthesizing machinery along actin filament bundles.

Indirect immunofluorescent microscopy was used to study the distribution of elongation factor 2 (eEF-2) in fixed human skin diploid and mouse embryo fibroblasts. It was found earlier that some of the eEF-2, ribosomes and initiation factor 2 (eIF-2) are co-localized with a part of the actin microfilament bundles in these cells (Gavrilova et al., 1987; Shestakova et al., 1991). Here it has been shown that inhibition of protein synthesis either by inactivation of eEF-2 itself with diphtheria toxin or by inactivation of ribosomes with ricin does not abolish the distribution of eEF-2 along the actin microfilament bundles. At the same time, the disassembly of actin microfilaments by cytochalasin D results also in the disappearance of eEF-2- carrying threads. This means that the eEF-2-carrying threads do not exist per se, and that the organization of eEF-2 in visible "filaments" depends upon the integrity of the actin cytoskeleton.

Actin Cytoskeleton↗
PubMed1993

Co-localization of components of the protein-synthesizing machinery with the cytoskeleton in G0-arrested cells.

The distribution of eukaryotic elongation factor 2 (eEF-2) in G0-arrested fixed human skin diploid fibroblasts was studied by indirect immunofluorescent microscopy. It was found earlier that the main part of eEF-2 in cycling cells was located near the nucleus in the endoplasm (Gavrilova et al., 1987). It has been demonstrated here that the transition from proliferation to the G0 phase of the cell cycle leads to the distribution of eEF-2 mainly along the intermediate filaments and/or microtubules. Both in cycling and in G0-arrested fibroblasts a portion of eEF-2 is also co-localized with actin microfilament bundles. The reversion of the cells from the G0 phase to proliferation is accompanied by rearrangement of the actin cytoskeleton and reversal to the original pattern of eEF-2 distribution. It is likely that the different types of cytoskeleton in eukaryotic cells can be involved in organization of protein-synthesizing machinery.

Actins↗
PubMed1991

Some of eukaryotic elongation factor 2 is colocalized with actin microfilament bundles in mouse embryo fibroblasts.

Indirect immunofluorescent microscopy was used to study the distribution of eukaryotic elongation factor 2 (EF-2) in cultured mouse embryo fibroblasts. The perinuclear area (endoplasm) of all the cells and many straight cables running along the whole cytoplasm were stained with monospecific goat or rabbit antibodies to rat liver EF-2. Double staining of the cells with antibodies to EF-2 and rhodaminyl-phalloidin (used for actin microfilament detection) showed that EF-2 containing cables coincided with bundles of actin microfilaments. Not all actin microfilament bundles contained EF-2: sometimes EF-2 was not observed in bundles running along the cell edges or in actin microfilament junctions. Triton X-100 extracted most of EF-2 from the cells and no actin microfilament bundles were stained with the EF-2 antibodies in the Triton-extracted cells. Thus, in mouse embryo fibroblasts EF-2 can be found along actin microfilament bundles, but it is unlikely to be their integral protein.

Actin Cytoskeleton↗
PubMed1989

[The effect of mutations in ribosomal proteins S4, S12 and L7/L12 on EF-Tu-dependent expenditure of GTP in the process of codon-specific elongation and misreading of poly(U)].

The effect of mutations in ribosomal proteins S4 (rpsD12), S12 (rpsL282) and L7/L12 (rplL265) of Escherichia coli K12 on the EF-Tu-dependent expenditure of GTP during codon-specific elongation (poly(Phe) synthesis on poly(U] and misreading (poly(Leu) synthesis on poly(U], was studied. Under the conditions used the mutations in proteins S4 and L7/L12 did not practically affect the EF-Tu-dependent expenditure of GTR during the poly(Phe) synthesis on poly(U): the GTP/Phe ratio was about 1, as in the case of the wild strain. Under the same conditions, the ribosomes with a mutant S12 protein tended to discard some amount of Phe-tRNA, as a result of which the GTP/Phe ratio increased to about 3. The marked inhibition of misreading by ribosomes with a mutant S12 protein was accompanied by a significant increase of GTP expenditure at the stage of EF-Tu-dependent non-cognate aminoacyl-tRNA binding. In mutant S 12 proteins the GTP/Leu ratio was about 30-40, whereas in the wild type it was about 12. In contrast, stimulation of misreading by ribosomes with mutant S4 and L7/L12 proteins was accompanied by a decrease of the EF-Tu-dependent expenditure of GTP by 2-3 GTP molecules per one Leu residue included into the peptide.

Codon↗
PubMed1987

Immunofluorescent localization of protein synthesis components in mouse embryo fibroblasts.

The distribution of initiation factor 2(eIF-2) and elongation factor 2(EF-2) in cultured mouse embryo fibroblasts was studied and compared with the distribution of ribosomes. We used immunofluorescence microscopy with monospecific antibodies to eIF-2, EF-2, and proteins S3a and S7 of the small ribosomal subunit. Ribosomes and factors eIF-2 and EF-2 were found mainly in the vicinity of the cell nucleus. This perinuclear zone coincides with the endoplasm - the central part of the cell containing numerous membraneous organelles and inclusions. Besides the perinuclear zone, small stained regions could be seen at the periphery of some cells. After treatment of the cells with Triton X-100 in a buffer conditions, that stabilizes the major cytoskeletal structures, some of the ribosomes, eIF-2, and EF-2 remained bound to the insoluble material. These components were found near the nucleus and some were located along the microfilament bundles.

Animals↗
PubMed1986

The excess GTP hydrolyzed during mistranslation is expended at the stage of EF-Tu-promoted binding of non-cognate aminoacyl-tRNA.

The system of translation of Sepharose-bound poly(U) in which all ribosomes are active in peptide elongation was used to determine the stoichiometry of GTP hydrolysis at the stage of EF-Tu-promoted aminoacyl-tRNA binding. The ratio of GTP hydrolyzed at this stage per peptide bond was assayed during codon-specific elongation (polyphenylalanine synthesis) and misreading (polyleucine synthesis). It was demonstrated directly that the excess GTP hydrolyzed during misreading [(1984) FEBS Letters 178, 283-287] is expended at the stage of Ef-Tu-promoted binding of non-cognate aminoacyl-tRNA.

Escherichia coli↗
PubMed1985

Effect of streptomycin on the stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system.

The technique of Sepharose-bound template translation has been used to estimate the stoichiometry of GTP hydrolysis during peptide elongation in the presence of streptomycin. The presence of streptomycin has been shown to have no great effect on the elongation rate and the stoichiometry of GTP hydrolysis during codon-specific peptide elongation in the poly(U)-directed translation system: the molar ratio of hydrolysed GTP to incorporated phenylalanine was about 2. At the same time streptomycin exerted a significant effect during misreading when a ribosome-bound peptide in the poly(U)-programmed system was elongated by leucine or isoleucine residues: the miselongation was stimulated and hence the ratio of hydrolysed GTP per peptide bond was strongly reduced, as compared with the excessive GTP hydrolysis which is characteristic of the misreading system in the absence of streptomycin [(1984) FEBS Lett. 178, 283-287]. The conclusion has been made that streptomycin blocks the stage of correction ('proof-reading') following GTP hydrolysis during EF-Tu-dependent aminoacyl-tRNA binding.

Escherichia coli↗
PubMed1984

Stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system. Determination of GTP/peptide bond ratios during codon-specific elongation and misreading.

The stoichiometry of GTP hydrolysis during peptide elongation in the processes of codon-specific translation and misreading of polyuridylic acid was determined in a cell-free system in which all ribosomes were active in peptide synthesis. Ribosomes carrying oligophenylalanine presynthesized on poly(U) covalently bound to Sepharose were used. In the codon-specific translation of poly(Phe) on poly(U)-Sepharose at optimal Mg2+ concentration (6 mM MgCl2), the ratio of GTP cleaved to Phe polymerized was found to be about 2 (+/- 0.1). Under the same conditions but during misreading (elongation of polyleucine on poly(U)-Sepharose) the GTP/Leu ratio increased 10 times (from 16 to 25 in different experiments).

Codon↗
PubMed1983

[Stoichiometry of GTP breakdown during peptide synthesis on the ribosome. Stoichiometry of GTP hydrolysis during elongation of polyphenylalanine on polyuridylic acid].

The stoichiometry of GTP hydrolysis during poly(Phe) elongation by Phe on poly(U) covalently bound to Sepharose was determined. The concentrations of both EF-T and EF-G were saturating. The GTP/Phe stoichiometry was calculated without the usual correction for the uncoupled ribosomal EF-T and EF-G dependent GTP hydrolysis. At the Mg2+ optimum (6 mM) for the poly(Phe) elongation on poly(U) . Sepharose the stoichiometry ratio of GTP/Phe was 1.9/2.1. This indicates that two (or less) GTP molecules coupled with poly(Phe) elongation by Phe on poly(U) . Sepharose are hydrolyzed.

Escherichia coli↗
PubMed1982

[Stoichiometry of GTP hydrolysis during peptide synthesis on the ribosome. GTP hydrolysis uncoupled with ribosomal peptide synthesis and dependent on preparation of elongation factor T].

It is shown that preparations of EF-T are practically free from GTPase activity, if they were formed from EF-Tu and EF-Ts carefully purified from GTPase beforehand. Some Ef-T-dependent GTPase can arise after addition of tRNA or aminoacyl-tRNA preparations to a EF-T+ribosomes mixture. Poly(U) stimulates, as a rule, EF-T-dependent GTPase in the EF-T/ribosomes+tRNA mixture. Uncoupled EF-T-dependent GTPase of the EF-T+ribosomes+tRNA+poly(U) mixture is by 1--2 orders of magnitude less than uncoupled EF-G-dependent GTPase under the same conditions. A conclusion is made that uncoupled EF-T-dependent GTPase is an inevitable obstacle when studying the GTP expenditure in the process of ribosomal protein synthesis, if a traditional cell-free protein-synthesizing system is used for this purpose.

Escherichia coli↗
PubMed1980

[Stoichiometry of GTP hydrolysis during peptide synthesis on the ribosome. I. Factor-independent GTPase and ATPase of ribosomal preparations].

It has been found that preparations of Escherichia coli (MRE-600) ribosomes can display GTPase and ATPase activities independent of elongation factors EF-Tu and EF-G. The GTPase and ATPase are localized on ribosomal 50S subparticles, whereas 30S subparticles are free of the activities and do not stimulate them upon association with the 50S subparticles to form complete ribosomes. The GTPase and ATPase can be removed from the ribosomes and their 50S subparticles by treatment with 1 M NH4Cl or 50% ethanol in the cold. Ribosomal preparations freed from the factor-independent GTPase and ATPase retain their basic functional features. The data obtained do not permit to solve finally whether the factor-independent GTPase and ATPase revealed are components of ribosomes or represent a contamination rather firmly bound to the ribosomes. However, in any case this finding can contribute to an uncoupled hydrolysis of GTP and should be considered when studying the stoichiometry of triphosphate expenditure in the process of ribosomal protein synthesis.

Adenosine Triphosphatases↗