Synthesis and study on activity in vitro of the high purity human butyrylcholinesterase conjugated with gold nanoparticles.

The aim of this research was to design a method of immobilization of high-purity human butyrylcholinesterase on the surface of gold nanoparticles preserving the activity of the enzyme. In order to achieve this aim, the method of fractionation and purification of human butyrylcholinesterase from plasma was modified. The synthesis of 15-nm gold nanoparticles was carried out by citrated method. A method of conjugation of the high-purity butyrylcholinesterase with gold nanoparticles was developed. It was found that the Immobilization of butyrylcholinesterase on the surface of gold nanoparticles resulted in a significant (to 23%) increase in the specific activity of the enzyme.

[A STUDY OF THE ISOLATED BACTERIOPHAGE ΦAB-SP7 ADSORPTION ON THE CELL SURFACE OF THE AZOSPIRILLUM BRASILENSE SP7].

The bacteriophage ΦAb-Sp7 was isolated from the cells of the Azospirillum brasilense Sp7. The morphology, size of the gram-negative colonies, and range of lytic activity against other strains and species of the genus Azospirillum was tested. The isolated phage DNA was examined using electrophoretic and restriction analysis, and the size of the genome were established. The electron microscopy. resuIts show that the phage (capsid) has a strand-like form. The electron microscopy study of the bacteriophage ΦAb-Sp7 adsorption on the A. brasilense Sp7 bacterial surface was performed.

[Determination of Azospirillum Brasilense Cells With Bacteriophages via Electrooptical Analysis of Microbial Suspensions].

The dependence-of changes in the electrooptical properties of Azospirillum brasilense cell suspension Sp7 during interaction with bacteriophage ΦAb-Sp7 on the number and time of interactions was studied. Incubation of cells with bacteriophage significantly changed the electrooptical signal within one minute. The selective effect of bacteriophage ΦAb on 18 strains of bacteria of the genus Azospirillum was studied: A. amazonense Ami4, A. brasilense Sp7, Cd, Sp107, Sp245, Jm6B2, Brl4, KR77, S17, S27, SR55, SR75, A. halopraeferans Au4, A. irakense KBC1, K A3, A. lipoferum Sp59b, SR65 and RG20a. We determined the limit of reliable determination of microbial cells infected with bacteriophage: - 10(4) cells/mL. The presence of foreign cell cultures of E. coli B-878 and E. coli XL-1 did not complicate the detection of A brasilense Sp7 cells with the use of bacteriophage ΦAb-Sp7. The results demonstrated that bacteriophage (ΦAb-Sp7 can be used for the detection of Azospirillum microbial cells via t electrooptical analysis of cell suspensions.

[Intracellular lectins of Lentinus edodes at various developmental stages of the fungus].

A number of lectins varying in polypeptide composition and carbohydrate specificity were isolated from Lentinus edodes at different stages of its morphogenesis: nonpigmented mycelium, brown mycelium film, and fruiting body. Three lectins were identified at the nonpigmented mycelium stage, two of them being dimers consisting of 16 and 45 kDa and 16 and 42 kDa subunits; the third is a tetramer of 16, 39, 42, and 45 kDa subunits. The fractions with lectin activity obtained at the brown mycelium film stage contained polypeptides of 24, 30, and 38 kDa, characteristic of this morphological structure. The fruiting body was shown to contain two lectins of 43 and 55 kDa. All of the isolated lectins expressed the highest affinity towards L,D-melibiose, D-lactose, and D-galactose.

Investigation of enzymatic degradation of pectin polysaccharides under limiting conditions.

The dynamics of changes in spectra of oligosaccharide fragments formed during enzymatic degradation of plant pectins at low enzyme/substrate ratio was studied. It is shown that degradation of deesterified pectin molecules is a discrete and determined process manifested in establishment of a stable polysaccharide spectrum. It is noted that introduction of chemical modifications into the polysaccharide substrate structure preserves the discreteness of the polymer molecule fragmentation but changes the spectrum of formed oligosaccharide fragments. It is supposed that degradation is defined by the spatial (three-dimensional) organization of the polysaccharide molecule.

Selection and characterization of phage miniantibodies to actins of different origin.

Single-chain miniantibodies (scFv's) to actin were obtained by the phage display method. A naive combinatorial phage display library of murine scFv's (containing 2x10(8) independent recombinant clones) was used to select miniantibodies. After three rounds of selection two clones producing miniantibodies to chicken smooth muscle actin with affinity constants of 1.4x10(7) and 1.2x10(6) M(-1) were chosen. The isolated miniantibodies could specifically detect various plant and animal actins.

[Use of colloid gold conjugates for identification of actins of various origin]. / Ispol'zovanie kon''ugatov kolloidnogo zolota dlia identifikatsii aktinov razlichnogo proiskhozhdeniia.

Some optimized methods of purifying actin from animal, plant, and bacterial cells were developed. Variants of the preparation of antiactin antibodies are described which use both traditional methods of immunization and phase display technology and antigen adsorption on colloidal gold particles. The conjugates of colloidal gold with phalloidin and heavy meromyosin as well as with antibodies were proposed. It was shown that these markers make it possible to reliably identify actins of different origin by the methods of light, and electron microscopy and dot-analysis.

Influence of divalent cations on the catalytic properties and secondary structure of unadenylylated glutamine synthetase from Azospirillum brasilense.

Fully unadenylylated glutamine synthetase (GS) from the endophytic bacterium Azospirillum brasilense Sp245 was isolated and purified. The enzyme was electrophoretically homogeneous and contained strongly bound metal ions, which could not be removed by dialysis. Mn2+, Mg2+, and Co2+ were found to be effective in supporting biosynthetic activity of the A. brasilense GS. Some kinetic properties of Mn2+-activated and Mg2+-activated unadenylylated GS were characterized. Circular dichroism analysis of the enzyme showed that the A. brasilense GS is a highly structured protein: 59% of its residues form alpha-helices and 13% beta-strands. Removal of the metal ions from the A. brasilense GS by treatment with EDTA resulted in alterations in the enzyme secondary structure.

[The detection and nature of the labelling of F-actin using a phalloidin-colloidal gold marker]. / Vyiavlenie i kharakter mecheniia F-aktina markerom falloidin--kolloidnoe zoloto.

On the basis of a conjugate of phalloidin-colloidal gold a marker has been obtained for electronmicroscopic identification and study of marking character of filament actins. Using this marker the filamentous form of actins from rabbit skeletal muscles and chicken gizzard was revealed. The marking characters of the two actins appeared to differ at the level of electron microscopy. A periodicity of the marker setting with a step of 105.4 +/- 1.7 nm on the rabbit F-actin was revealed. A possibility of differential evaluation of the filamentous and globular forms of the actins was shown when using the same marker in dot-blot analysis procedure with the sensitivity from 15 to 50 ng.