Biomedical subjects

L A Baratova

Publications and source records attributed to L A Baratova.

At least 19 recordsLinked to original sources

PubMed2006

A comparative study of functional properties of calf chymosin and its recombinant forms.

The action of calf chymosin obtained from transgenic sheep milk and the recombinant protein expressed in yeast Kluyveromyces lactis (Maxiren) on fluorogenic peptide substrates, namely Abz-A-A-F-F-A-A-Ded, Abz-A-A-F-F-A-A-pNA, Abz-A-F-F-A-A-Ded, Abz-A-A-F-F-A-Ded, Abz-A-A-F-F-Ded, Abz-A-A-F-F-pNA, and heptapeptide L-S-F-M-A-I-P-NH2, a fragment of kappa-casein (the native chymosin substrate), was investigated. It has been established that transgenic chymosin and recombinant chymosin (Maxiren) differ from the native enzyme in their action on low molecular weight substrates, whereas there was no difference in enzymatic action on protein substrates. Pepstatin, a specific inhibitor of aspartic proteinases, inhibits the recombinant chymosin forms less efficiently than the native enzyme. Perhaps this is associated with local conformational changes in the substrate binding site of recombinant chymosin occurring during the formation of the protein globule.

Amino Acid Sequence↗

PubMed2003

Dysfunctionality of a tobacco mosaic virus movement protein mutant mimicking threonine 104 phosphorylation.

Replication of tobacco mosaic virus (TMV) is connected with endoplasmic reticulum (ER)-associated membranes at early stages of infection. This study reports that TMV movement protein (MP)-specific protein kinases (PKs) associated with the ER of tobacco were capable of phosphorylating Thr(104) in TMV MP. The MP-specific PKs with apparent molecular masses of about 45-50 kDa and 38 kDa were revealed by gel PK assays. Two types of mutations were introduced in TMV MP gene of wild-type TMV U1 genome to substitute Thr(104) by neutral Ala or by negatively charged Asp. Mutation of Thr(104) to Ala did not affect the size of necrotic lesions induced by the mutant virus in Nicotiana tabacum Xanthi nc. plants. Conversely, mutation of Thr to Asp mimicking Thr(104) phosphorylation strongly inhibited cell-to-cell movement. The possible role of Thr(104) phosphorylation in TMV MP function is discussed.

Amino Acid Substitution↗

PubMed2002

Studying the spatial organization of membrane proteins by means of tritium stratigraphy: bacteriorhodopsin in purple membrane.

The topography of bacteriorhodopsin (bR) in situ was earlier studied by using the tritium bombardment approach [Eur. J. Biochem. 178 (1988) 123]. Now, having the X-ray crystallography data of bR at atom resolution [Proc. Natl. Acad. Sci. 95 (1998) 11673], we estimated the influence of membrane environment (lipid and protein) on tritium incorporation into amino acid residues forming transmembrane helices. We have determined the tritium flux attenuation coefficients for residues 10-29 of helix A. They turned out to be low (0.04+/-0.02 A(-1)) for residues adjacent to the lipid matrix, and almost fourfold higher (0.15+/-0.05 A(-1)) for those oriented to the neighboring transmembrane helices. We believe that tritium incorporation data could help modeling transmembrane segment arrangement in the membrane.

Bacteriorhodopsins↗

PubMed2001

Studying liposomes by tritium bombardment.

Bilayer liposomes from a mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPC: DPPE = 8:2, molar ratio) or DPPC labeled with 14C-DPPC (DPPC: 14C-DPPC) were bombarded with thermally activated tritium atoms. The tritiated liposomes were hydrolyzed by phospholipase C, and the tritium incorporation into different parts of the bilayer along its thickness was determined. The tritium flux attenuation coefficients were calculated for the headgroup (k1 = 0.176+/-0.032 A(-1)) and acylglycerol residue (k2 = 0.046+/-0.004 A(-1)) layers indicating a preferential attenuation of the tritium flux in the headgroup region and relative transparence of the membrane hydrophobic part. The finding is potentially important to apply tritium bombardment for investigation of spatial organization of transmembrane proteins in their native lipid environment.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2001

In situ spatial organization of Potato virus A coat protein subunits as assessed by tritium bombardment.

Potato virus A (PVA) particles were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the label in the amino acids of the coat protein was determined to assess their in situ steric accessibility. This method revealed that the N-terminal 15 amino acids of the PVA coat protein and a region comprising amino acids 27 to 50 are the most accessible at the particle surface to labeling with tritium atoms. A model of the spatial arrangement of the PVA coat protein polypeptide chain within the virus particle was derived from the experimental data obtained by tritium bombardment combined with predictions of secondary-structure elements and the principles of packing alpha-helices and beta-structures in proteins. The model predicts three regions of tertiary structure: (i) the surface-exposed N-terminal region, comprising an unstructured N terminus of 8 amino acids and two beta-strands, (ii) a C-terminal region including two alpha-helices, as well as three beta-strands that form a two-layer structure called an abCd unit, and (iii) a central region comprising a bundle of four alpha-helices in a fold similar to that found in tobacco mosaic virus coat protein. This is the first model of the three-dimensional structure of a potyvirus coat protein.

Capsid↗

PubMed2000

Determination of biologically active low-molecular-mass thiols in human blood. II. High-performance capillary electrophoresis with photometric detection.

A new high-performance capillary electrophoresis assay for aminothiols in human blood, including homocysteine, a marker of several human metabolism disorders, has been developed. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with sulfosalicylic acid, and conjugation of the thiols with monobromobimane. Derivatized thiols were separated in a sodium phosphate buffer using a fused-silica capillary (65 cm x 50 microm I.D.) at 30 degrees C. With the electric field of 250 V cm(-1), separation of homocysteine, glutathione and cysteine occurred at less than 10 min. Detection at 250 or 234 nm was used to confirm the monobimane-thiols peaks. The detection limit was approximately 5 nmol/ml for all labeled aminothiols. The proposed method for these compounds' analysis included simple sample preparation, high selectivity, good linearity (r2>0.999), high reproducibility (within-run precision for derivatized aminothiol peaks area RSD<5% for three times consequently injected sample); high reliability and the small volumes required for analysis made it suitable for clinical studies.

Electrophoresis, Capillary↗

PubMed2000

Qualitative and quantitative determination of biologically active low-molecular-mass thiols in human blood by reversed-phase high-performance liquid chromatography with photometry and fluorescence detection.

The reversed-phase high-performance liquid chromatographic method employing photometry and fluorescence detection is described for the precise reproducible simultaneous measurement of total homocysteine (tHcy), cysteine (Cys), and glutathione (GSH) in human blood. Sample preparation involves conversion of disulfides to free thiols with triphenylphosphine, precipitation of proteins with trichloroacetic acid, conjugation of the thiols with monobromobimane (mBrB). The aminothiol assay is optimized by reduction and derivatization step conditions (pH, temperature and time of reactions) to obtain reliable quantitative results within the concentration range corresponding to normal and pathological levels of these thiols in human blood.

Chromatography, High Pressure Liquid↗

PubMed1999

The in situ spatial arrangement of the influenza A virus matrix protein M1 assessed by tritium bombardment.

Intact influenza A virions were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the label in the matrix protein M1 was analyzed to determine the in situ accessibility of its tryptic fragments. These data were combined with the previously reported x-ray crystal structure of the M1 fragment 2-158 [Sha, B. & Luo, M. (1997) Nat. Struct. Biol. 4, 239-244] and the predicted topology of the C domain (159-252) to propose a model of M1 arrangement in the virus particle.

Animals↗

PubMed1998

Tritium planigraphy: from the accessible surface to the spatial structure of a protein.

The method of tritium planigraphy, which provides comprehensive information on the accessible surface of macromolecules, allows an attempt at reconstructing the three-dimensional structure of a protein from the experimental data on residue accessibility for labeling. The semiempirical algorithm proposed for globular proteins involves (i) predicting theoretically the secondary structure elements (SSEs), (ii) experimentally determining the residue-accessibility profile by bombarding the whole protein with a beam of hot tritium atoms, (iii) generating the residue-accessibility profiles for isolated SSEs by computer simulation, (iv) locating the contacts between SSEs by collating the experimental and simulated accessibility profiles, and (v) assembling the SSEs into a compact model via these contact regions in accordance with certain rules. For sperm whale myoglobin, carp and pike parvalbumins, the lambda cro repressor, and hen egg lysozyme, this algorithm yields the most realistic models when SSEs are assembled sequentially from the amino to the carboxyl end of the protein chain.

Algorithms↗

PubMed1998

Covalent chromatography of influenza virus membrane M1 protein on activated thiopropyl Sepharose-6B.

The M1 protein of influenza virus is a highly hydrophobic polypeptide that is resistant to enzyme cleavage during incubation in water solutions. We show here that the M1 protein that is immobilized on an insoluble activated support (thiopropyl Sepharose-6B) by means of a thiol-disulfide exchange reaction acquires sensitivity to trypsin. After tryptic digestion noncysteine-containing peptides of M1 were removed by washing the support, while cysteine-containing ones were detached from the support by reduction. As a result, 24 unique tryptic peptides of M1 protein were clearly separated by reversed-phase high-performance liquid chromatography. The described method opens a new way to the investigation of functional properties of distinct domains of viral thiol proteins.

Amino Acid Sequence↗

PubMed1996

[Isolation and properties of serine proteinase PC from the Kamchatka crab, Paralithodes camtschatica--a proteolytic enzyme with broad specificity].

A homogeneous serine proteinase PC has been isolated from the Camchatka crab (Paralithodes camtschatica) hepatopancreas using affinity chromatography on arginine-Sepharose, protamine tryptic peptide-agarose and ion-exchange chromatography on Mono-Q, with a 68% yield. The enzyme is completely inhibited by diisopropylfluorophosphate, a typical inhibitor for serine proteinases. The molecular mass of the proteinase is 29 kDa, pI is 3.0. The proteinase splits Glp-Phe-Ala-pNA optimally at pH 7.5 and 47-55 degrees C; Km is 0.83 mM, kcat is 67 s-1. The enzyme is stable at pH 4-9. Proteinase PC possesses a broad substrate specificity and splits the peptide bonds formed by the carboxyl group of hydrophobic amino acids, arginine and lysine, in peptides and proteins. The enzyme hydrolyzes fibrin and collagen. Its N-terminal sequence, IVGGQEATP, reveals a 90% homology with analogous sequences of collagenolytic proteinases from other crab species.

Amino Acid Sequence↗

PubMed1992

The organization of potato virus X coat proteins in virus particles studied by tritium planigraphy and model building.

Potato virus X particles containing the intact, undegraded Ps form of the coat protein and particles containing the in situ degraded Pf form of the coat protein, which is devoid of 19-21 amino acids from the N-terminus, were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the tritium label was studied. The tritium planigraphy revealed that the N-terminal region of the coat protein is the most accessible region for both type of PVX particles. The C-terminal region of the coat protein in the intact virus particles is almost inaccessible to the hot tritium atoms, whereas in Pf particles this region becomes available for the tritium label. A model of PVX coat protein tertiary structure was built, taking into account the predicted secondary structure of the protein, the principles of packing alpha-helices and beta-structure in globular proteins, and known biochemical, immunological, and tritium bombardment data. In the model one beta-sheet consisting of beta-strands at regions 1-12, 14-22, and 24-33 flanks the molecule and forms the outside surface of the PVX particles.

Amino Acid Sequence↗

PubMed1992

The topography of the surface of potato virus X: tritium planigraphy and immunological analysis.

Thermally activated tritium atoms were used to probe the surface topography of the coat protein of potato virus X (PVX) potexvirus. The accessibility profile of amino acid residues in the polypeptide chain was determined from data on the intramolecular distribution of tritium label in the PVX coat protein. Tryptic peptides T1 and T2, as well as parts of peptides T3 and T5, from the PVX particles were all located in the N-terminal region of the PVX coat protein and were accessible to tritium labelling, whereas the C-terminal region of the coat protein was practically inaccessible to it. Indirect ELISA and immunoblotting with two PVX-specific monoclonal antibodies confirmed that the N terminus of the coat protein (residues 1 to 56) was exposed on the virus surface, and furthermore that this region forms a highly immunogenic virus-specific antigenic region. The data obtained support the spatial model of PVX, in which the N-terminal amino acids of the coat protein are exposed at the particle surface, and the C-terminal region is buried in the particle. The spatial organization of the PVX coat proteins differs from the model proposed for other filamentous plant viruses such as potyviruses and tobamoviruses where both the N and C termini of the coat protein are located at the particles' surface.

Amino Acid Sequence↗

PubMed1991

A model for the study of the mechanism of a low pH-induced interaction of the virus fusion proteins and cell membranes.

A model is proposed for the study of molecular mechanisms of a low pH-induced interaction of fusion proteins of enveloped viruses and cell membranes. The model consists of large monolamellar liposomes containing ionophore nigericin in their membranes and ectodomains of fusion protein in their inner space. The process of interaction of the protein with the lipid bilayer is triggered by acidification of the liposomal constituents to the pH of fusion with the help of nigericin by adding citric acid to the outer medium. To visualize the protein structural reorganization, the tritium planigraphy was used. Comparison of the values of specific labelling of the proteins and distribution of radioactivity in individual amino acids in control (at neutral pH) and experimental liposome samples (at the pH of fusion) permits to realise the character of protein-membrane interaction. We have obtained the first results in the study of interaction of the bromelain-released soluble ectodomain of the HAXX molecule (BHA)--with the lipid membrane. The observed increase in the protein specific activity and selective increase in the specific activity of hydrophobic amino acids Ile, Phe and Tyr in experimental liposome samples as compared with the controls did not contradict to the conventional concept, that a hydrophobic N-terminus of HA2 subunit of hemagglutinin is responsible for its interaction with lipid membranes.

Hemagglutinin Glycoproteins, Influenza Virus↗

PubMed1990

Prediction of arenavirus fusion peptides on the basis of computer analysis of envelope protein sequences.

Theoretical search and selection criteria for putative fusion peptides of enveloped viruses are proposed. Arenavirus fusion peptides are predicted on the basis of computer-assisted analysis of amino acid sequences of arenavirus envelope proteins and elements of their secondary and tertiary structure. Accordingly, two regions of GP2 surface protein from 5 viruses of Arenaviridae family have been detected with properties typical of fusion peptides of other enveloped viruses. One region, named peptide IV, located at the N-terminus of the GP2 protein, is followed by the other region or peptide V, more likely candidate for the arenavirus fusion peptide.

Amino Acid Sequence↗

PubMed1990

[A comparative study of phenylalanyl-tRNA synthetases from Escherichia coli and Thermus thermophilus by the tritium topography method].

A comparative study of thermostability and amino acid composition of phenylalanyl-tRNA synthetases from E. coli and Thermus thermophilus HB8 has been carried out. In the thermophilic protein the proline, leucine, phenylalanine, arginine content was considerably increased, whereas that of asparagine, isoleucine, serine, threonine and lysine was decreased as compared to the mesophilic protein. Using tritium topography, Pro, (Leu + Ile) and Gly were found to be the most accessible on the surfaces of the both enzymes. In the E. coli enzyme the threonine residues were also easy to access, while on the surface of the thermophilic enzyme arginine residues were more abundant. A quantitative assay of the surface compositions revealed the increased exposure of (Leu + Ile) residues in the thermophilic protein as well as of the charged asparagine and arginine residues. A possible relationship of the observed effects to thermostability is discussed.

Enzyme Stability↗

PubMed1990

Comparative study of the phenylalanyl-tRNA synthetases from Escherichia coli and Thermus thermophlus by the tritium topography method.

A comparative study of thermostability and aminoacid composition of the phenylalanyl-tRNA synthetases from E. coli and Thermus thermophilus HB8 has been carried out. Compared with the mesophilic enzyme, a considerable increase of Pro, Leu, Phe, Arg and decrease of Asx, Ile, Ser, Thr and Lys content have been revealed in the thermophilic protein. Using tritium topography, Pro, (Leu + Ile) and Gly were found to be the most accessible on the surfaces of both the enzymes. In the E. coli enzyme, Thr residues were also easy to access while on the surface of the thermophilic enzyme there were more Arg residues. The quantitative assay of the surface compositions revealed the increased exposure of the (Leu + Ile) residues on the thermophilic protein as well as of the charged Asx and Arg residues. A possible correlation of the observed effects with thermostability is discussed.

Amino Acids↗

PubMed1989

[The amino acids of the blood in patients with peritonitis: the significance of Fisher's index].

Blood plasma amino acid levels in 10 patients with peritonitis are analyzed. Nonsurvivors, in contrast to survivors, had severe amino acid derangements. Aminoacidemia and amino acid imbalance were registered early in the course of the illness. Increased concentrations of aromatic amino acids: phenylalanine and tyrosine, and low concentrations of branched-chain amino acids: valine, leucine, and isoleucine were associated with a decrease of Fisher's index (BCAA/AAA molar ratio) from 3.0 +/- 0.5 (normal) to 2.0 +/- 0.5 which was of high diagnostic and prognostic significance. The nature of such imbalance and its role in protein derangements are discussed.

Adult↗