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Biomedical subjects

A A Shul'ga

Publications and source records attributed to A A Shul'ga.

At least 19 recordsLinked to original sources
PubMed2000

[Ribonuclease from Bacillus thuringiensis var. subtoxicus. Gene structure and biosynthesis regulation].

The gene for extracellular guanyl-specific ribonuclease of Bacillus thuringiensis var. subtoxicus (RNase Bth), a close homologue of the B. intermedius RNase (binase), was completely sequenced. Analysis of nucleotide sequences in the regions adjoining RNase genes revealed an identical organization of the chromosomal loci of RNase Bth and binase. Growth characteristics of the Bacillus thuringiensis var. subtoxicus strain and its synthesis of RNase were studied. It was shown that the exogenous inorganic phosphate inhibits the biosynthesis of RNase. At the same time, actinomycin D in low doses stimulates the enzyme synthesis. Comparative analysis of the influence of inorganic phosphate and actinomycin D on the biosynthesis of RNAse Bth and binase suggests a possibility of coincidence of regulatory pathways of synthesis of these enzymes.

Amino Acid Sequence↗
PubMed1997

[Secondary structure of binase in solution by 1H NMR].

Nearly all resonances were assigned in the two-dimensional 1H NMR spectra of binase, guanylospecific ribonuclease from Bacillus intermedius containing 109 amino acid residues. The exchange rates of amide protons with the solvent deuterium were measured in 2H2O at pH 6.7 and 30 degrees C. Coupling constants 3J of H-NC alpha-H, NOE contacts, solvent exchange rates of amide protons, and indices of C alpha H chemical shifts were measured, and the binase secondary structure was deduced from these data. It involves three alpha-helices in the N-terminal part (the 6-16, 26-31, and 41-45 segments) and a beta-sheet formed by five antiparallel beta-strands (51-55, 71-75, 86-90, 95-99, and 104-108 segments). The binase secondary structure was compared with that of its closest homologue, barnase from B. amyloliquefaciens.

Amino Acid Sequence↗
PubMed1994

Glucose-sensitive enzyme field effect transistor using potassium ferricyanide as an oxidizing substrate.

A glucose-sensitive field effect transistor was fabricated by immobilizing glucose oxidase on the gate of a pH-sensitive field effect transistor. Calibration curves of the biosensor were measured in phosphate and TRIS buffers in the presence of potassium ferricyanide. The use of the latter as an oxidizing substrate in the biocatalytic oxidation of glucose leads to an increase of the acidification rate of the solution inside the enzymatic layer, because three protons are now generated per one molecule of glucose instead of only one when the natural oxidizing cosubstrate, oxygen, is used. Depending on the concentration of ferricyanide we observe a 10-100 times increase of the biosensor response in concentrated buffer solutions and a substantial extension of its dynamic range. At sufficiently high concentrations of ferricyanide, the calibration curves in both buffers have a sigmoidal shape in linear coordinates with local pH changes on the surface of the field effect transistor reaching about two pH units in the saturation range. The resulting saturation of the curves at higher glucose concentrations is due to the inhibition of the activity of glucose oxidase at acidic pH by Cl- ions present in the solution. The proposed approach may be extended to allow the detection of a wide range of analytes using enzyme field effect transistors based on the enzymes for which reoxidation of the cofactor (coenzyme) leads to a liberation of H+ ions.

Biosensing Techniques↗
PubMed1994

Thin-film conductometric biosensors for glucose and urea determination.

The characteristics of the developed conductometric biosensors for urea and glucose determination are described. Conductometric transducers based on thin-film interdigitated metal (Au, Cr, Cu, Ni) electrodes were studied, and enzymes urease and glucose oxidase were used for the selective membranes formation on the chips having gold electrodes. The influence of ionic strength and buffer capacity of the samples on the biosensors response in kinetic and steady-state modes of measurements was thoroughly tested. It was shown that the kinetic response of the sensors does not depend on the buffer capacity of the analyzed sample. In basic features the performance of the developed biosensors is rather close to that of respective enzyme field effect transistor, though the former are much superior when the technological complexity of the transducer itself is considered and taking into account that conductometric sensors require no reference electrode.

Biosensing Techniques↗
PubMed1993

A cell biosensor specific for formaldehyde based on pH-sensitive transistors coupled to methylotrophic yeast cells with genetically adjusted metabolism.

A cell biosensor specific for formaldehyde was developed using double-mutant cells of the methylotrophic yeast Hansenula polymorpha A3-11. The activities of some of the enzymes in the metabolic pathway of the wild-strain cells were deliberately suppressed by introducing respective genetic blocks to optimize the selectivity and acidification rate. Mutant yeast cells produced in this way were immobilized in Ca-alginate gel on the gate of a pH-sensitive field effect transistor. The local acidification of the extracellular medium due to specific conversion of formaldehyde was recorded. The steady-state response time of the biosensor was 2-3 min, i.e., about 10 times shorter than the response time for the alcohol-specific cell biosensors described earlier. The linear dynamic range of the sensor's response corresponds to formaldehyde concentrations of 2 to 200 mM. The operational stability of the sensor was not less than 4 h. The biosensor demonstrated high specificity to formaldehyde with no response to several organic acids, methanol, and other alcohols, except for low sensitivity to ethanol. The influence of sample buffer capacity and pH on the sensor response, as well as thermostability, was investigated.

Biosensing Techniques↗
PubMedDate not supplied

[The study of the metal-binding region in human growth hormone using immobilized metal ion affinity gel-electrophoresis].

The zinc(II)-binding affinities of recombinant human growth hormone and two its mutants, 14-33 and 14-95, were studied using Immobilized Metal Ion Affinity Gel-electrophoresis (IMAG). The mutant hormones, composed of polypeptide chain segments of the human and porcine growth hormones, lacked His18, which may be crucial for binding of the intact hormone to the transition metal ions. The mutations did not affect the affinity of human growth hormone to immobilized zinc ions; the structural analysis implied that the human growth hormone contains two IDA-Zn(II) potential sorption sites formed by amino acid residues His21, Asp171, and Glu174 and/or His18 and Glu174.

Animals↗
PubMedDate not supplied

[I87E mutation prevents barstar dimerization].

The C40,82A;I87E mutant of barstar, an intracellular inhibitor of the ribonuclease barnase from Bacillus amyloliquefaciens, was obtained, and its physicochemical properties were studied. It was produced as a fusion protein with thioredoxin and then cleaved from this by EKmax enterokinase. The mutant was shown by NMR to retain the spatial structure of the wild-type protein but, in contrast to barstar, does not form the homodimers characteristic of barstar in aqueous solution. The mutant protein binds barnase with the dissociation constant (6.6 +/- 1.1) x 10(-11) M and exhibits other physicochemical properties similar to those of the wild-type barstar. This allows the use of C40,82A;I87E mutant instead of wild-type barstar in investigations where the protein dimerization is undesirable. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

Bacillus↗
PubMedDate not supplied

[Residual dipolar couplings and molecular dynamic calculations as a source for refinement of protein spatial structures].

The precision of techniques and factors affecting the interpretation of residual dipolar couplings (RDCs) in analysis of spatial structures of partially aligned proteins are discussed. Experimental RDC values were obtained for pairs of 1H-15N nuclei of the protein barstar partially aligned in a liquid crystalline matrix of bicelles composed of dimiristoylphosphatidylcholine and dihexanoylphosphatidylcholine. The observed couplings agree well with the spatial structures of barstar determined earlier by X-ray and NMR methods. However, the differences between the experimental and calculated RDCs that were calculated on the basis of the known spatial structures of barstar, exceed the experimental errors three- to fourfold. These discrepancies can be explained by differences in the protein structures in solution and in crystal, a limited precision of the X-ray analysis, and the intramolecular mobility of the protein molecule. A comparison of the results of modeling of the molecular dynamics of barstar in solution, crystal structures, and the experimental RDCs showed that the methods of molecular dynamics provide for a reasonable description of the character and amplitudes of internal motions and they should be considered for the correct determination of protein spatial structures from NMR spectroscopic data.

Amino Acid Substitution↗
PubMedDate not supplied

[Theoretical analysis of the amino acid sequence of receptors for growth hormones and prolactins. Prediction of ligand-binding segments].

At present the growth hormone and prolactin receptors were cloned along with their variant forms from human, rat, mouse, rabbit, bovine and sheep tissues. The functional topography of receptors is practically unknown. Because of the high price and difficulty of protein's total mutagenesis, it is reasonable to carry on a theoretical analysis of structures of receptors to predict the most probable ligand-binding sites. We studied the primary structures of known prolactin and growth hormone receptors using theoretical methods proved to be powerful in earlier structure--activity relationship investigations. We analyzed the secondary structure, conservative positions, hydrophilicity profiles of the growth hormone and prolactin receptors, and used the original method based on information theory to predict the sites which are promising for mutagenesis or peptide synthesis as probable ligand-binding sites. Three segments corresponding to the main conservative, hydrophilic and rare sites were predicted to form the ligand-binding determinant.

Amino Acid Sequence↗
PubMedDate not supplied

[Expression of neurotoxin II from Naja oxiana cobra venom in Escherichia coli in a hybrid form with thioredoxin].

Neurotoxin II from the venom of cobra Naja oxiana is a short type alpha-neurotoxin, which competitively inhibits nicotinic acetylcholine receptor. The toxin gene was expressed as a construct fused with the thioredoxin gene and the linker encoding the enteropeptidase recognition site and a Met residue between the genes. The fusion protein was mainly cleaved by cyanogen bromide, since enteropeptidase was less effective. The yield of neurotoxin II was 6 mg/l of the bacterial culture. The resulting recombinant protein was identified with native neurotoxin II by its N-terminal analysis, mass spectrometry, and NMR spectroscopy. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.

Animals↗
PubMedDate not supplied

[Amperometric enzyme biosensor with a glucose oxidase-polyaniline membrane].

An amperometric glucose biosensor was made by electrochemical polymerization of aniline onto the gold electrodes in presence of the enzyme glucose oxidase in the phosphate buffer solution with pH 7.0. Aniline is easily polymerized forming a thin film, which adheres tightly on the electrodes surface. During the electropolymerization process glucose oxidase was entrapped into polyaniline film which then became the catalyst of the enzyme reaction of glucose hydrolysis. Experiments were performed to determine optimal conditions of polyaniline-glucose oxidase film preparation. Glucose was amperometrically determined with the electrochemically fabricated biosensor in the concentration range 10(-4) M to 2 x 10(-2) M. The linearity of the enzyme electrode response ranged from 2 x 10(-4) M to 6 x 10(-3) M. The electrochemical synthesis of a polyaniline-enzyme thin film a high-technologic one and this permits fabricating various microbiosensors and multisensors in the continuous technological cycle.

Aniline Compounds↗
PubMedDate not supplied

[Use of conductometric microsensors for studying kinetic parameters of enzymes].

An alternative method based on thin-film conductometric microsensor is suggested for studying enzyme kinetics. It is established that the immobilized enzyme as well as soluble one is described by the classic lows of enzyme kinetics. Major parameters (Km and Vmax) are identical to those measured by the widely used methods and their amounts are 1.33 mM and 9.04 microS/min for soluble urease and 3.73 mM and 13.8 microS/min for immobilized urease; 5.26 mM and 13.9 microS/min for immobilized glucose oxidase; 13.6 mM and 122.0 microS/min for immobilized acetyl cholinesterase; 10.8 mM and 129.9 microS/min for immobilized butyryl cholinesterase. The possibility of conductometric analyzer application for detection of toxin concentration, in particular pesticides, is shown. Advantages and disadvantages of the system suggested are discussed.

Biosensing Techniques↗