[The thermophilic bacteria hydrolyzing agar: characterization of thermostable agarase].

Three strains of anaerobic thermophilic bacteria capable of growing on agarose as a source of energy and carbon were isolated from hot springs near Lake Baikal (Barguzin National Park) and the caldera Uzon (Kamchatka). Cells of all the three strains were spore bacilli with peritrichous flagellation. These isolates grew at a temperature of 55-60 degrees C and pH 6.5-7.0 and fermented a wide range of organic substrates. Analysis of the 16S rRNA sequences allowed us to ascribe the strains B5 and K14 to the genus Thermoanaerobacter and the strain K67 to the genus Caldoanaerobacter. According to the results of DNA-DNA hybridization, B5 was determined as belonging to the species Thermoanaerobacter wiegelii. Agarase was isolated by preparative PAGE and subsequent gel chromatography from the culture liquid of strain B5 grown on the medium containing 0.5% agarose and 0.3% galactose. The molecular weight of this enzyme amounted to 67 kDa and pI, to 4.2. The T. wiegelii B5 agarase was active in the pH range of 3.5 to 7.0 (optimum, 5.2) and temperature range of 50 to 80 degrees C (optimum, 70 degrees C). The preincubation of this enzyme at 90 degrees C for 60 min did not reduce the agarase activity. This activity increased in the presence of metal ions; the maximal effect was observed in the presence of 5 mM Mg2+ and 25 mM Co2+.

[Anticoagulant activity of low-molecular-weight sulfated derivatives of galactomannan from Cyamopsis tetragonoloba (L.) seeds].

Galactomannan from seeds of Cyamopsis tetragonoloba (L.) Taub. (guar) was depolymerized using immobilized enzymatic preparation celloviridin. A set of fragments whose molecular weights varied from 12.6 to 245.6 kDa was obtained. Sulfated derivatives of components of all fractions were synthesized, in which the content of HSO3(-) groups was 48.05% +/- 2.31. All preparations exhibited anticoagulant activity, which was recorded in vitro in two tests--aIIa and aXa. The antithrombin activity (aIIa) was high (up to 65-87 U/mg) and did not depend on the molecular weight of a sulfated derivative; in the second test (aXa), the effect of molecular weight was observed. Biospecific electrophoresis allowed us to detect the ability of galactomannan sulfates to form complexes with protamine sulfate, a classic antidote to heparin.

[Effect on low-molecular-weight heparin obtained using a chitinolytic complex on the anticoagulant activity of plasma in rabbits and rats].

The anticoagulant activity of low-molecular-weight heparin with an average molecular weight of 4.7 kD (LMWH-4.7) has been studied. This derivative was prepared from unfractionated heparin with the help of chitinolytic enzyme complex from Streptomyces kurssanovii. The antithrombin activity of LMWH-4.7 (aIla activity) was 72 +/- 9 IU/mg and the activity with respect to the blood coagulation factor Xa (aXa activity) was 200 +/- 33 IU/mg, which corresponded to an aXa/aIIa ratio of 2.8 (necessary for effective antithrombotic drugs). The aIIa and aXa activity exhibited a dose-dependent variation upon intravenous and subcutaneous injections in rabbits, so that a high aIIa/aXa ratio was retained: 5 min after the intravenous injection of a minimum dose (0.3 mg/kg), this ratio was 2, 7, and for a greater dose (3.0 mg/kg) it reached 3.8. Subcutaneous injections were followed by slow elimination of the anticoagulant within 24 h. LMWH-4.7 upon intraperitoneal injections produced a dose-dependent inhibition of a model thrombosis in rats. Complete inhibition was observed for a dose of 3 mg/kg. Thus, it is possible to obtain active LMW heparin with the help of chitinases.

[Anticoagulant activity of low-molecular-weight heparins obtained using a hydrolase complex].

The anticoagulant activity of low-molecular weight heparins (LMWH-PC) with average distribution of molecular weights within 3.4-5.8 kD was investigated. The samples of LMWH-PC were obtained from unfractionated heparin using immobilized enzyme complex of protease C. The LMWH-PC derivatives inhibited the activity of blood coagulation factors IIa (thrombin) and Xa. The LMWH-PC derivatives had an anti-factor-Xa activity up to 131-208 IU/mg and anti-factor-IIa activity up to 81-175 IU/mg. All LMWH-PC derivatives form complexes with protamine sulfate during electrophoresis in agarose gel. The anticoagulant activity of rabbit plasma exhibits a doze-dependent increase upon the intravenous or subcutaneous injection of LMWH-PC with a molecular weight of 5.4 kD.

[The determination of activity of low-molecular heparins against activated factor Xa by means of the diagnostic kit "Reachrom-Heparin" (scientific-production association "Renam")].

It is possible to successfully determine both the activity against activated factor Xa in animals plasma after heparins administration and specific activity against activated factor Xa of new anticoagulants by means of the domestic diagnostic kit "Reachrom-Heparin" developed by the company "Renam". It was compared to the similar kit "Berichrom heparin" (Dade Behring). No statistically significant differences were detected.

[Anticoagulant activity of sulfated polysaccharides].

The paper reviews published data on the chemical structure of sulfated polysaccharides of animal, plant, and microbial origin as well as synthetic or semi-synthetic derivatives, and on the anticoagulant activity of these compounds in vitro and in vivo. The influence of sulfated polysaccharides on the plasma and cell hemostasis links is considered. Relationships between the structure and activity and the mechanisms of action are discussed.

Ultrastructural study of chitosan effects on Klebsiella and staphylococci.

Antibacterial effect of chitosan on the morphofunctional organization of clinical strains of Klebsiella pneumoniae and Staphylococcus aureus was studied by transmission electron microscopy. Chitosan promoted aggregation of bacterial cells and disorganization of bacterial cell wall and cytoplasmic membrane, which leads to the release of bacterial contents into the environment. These structural changes result in bacterial death.

[Medical-prophylactic properties of the low-molecular-weight chitosan at environmental radiation injury]. / Lechebno-profilakticheskie svoistva nizkomolekuliarnogo khitozana pri éksperimental'nom luchevom porazhenii.

The possibility of the successful modification of radiation injury by chitosan with low molecular weight (10 kDa) has been established under experimental conditions. The survival of mice increased up to 72.7 and 44.7% respectively at intravenous and intramuscular injection 30 min before gamma-irradiation with a dose 8 Gy (LD97). In guinea pigs the effect was 50-52.6% at intravenous and 40% at intramuscular administration 1-3 h after irradiation with a dose 5 Gy (LD90). Radioprotective efficiency of 10 kDa chitosan is close to that of high-molecular-weight (65-70 kDa) preceding (medicine RS-10 and RS-11).

[Hydrolysis of heparin by the immobilized enzymatic complex from Streptomyces kurssanovii]. / Gidroliz geparina immobilizovannym fermentnym kompleksom iz Streptomyces kurssanovii.

The possibility of obtaining low-molecular-weight heparins using the chitinolytic enzymatic complex immobilized on Silochrom has been demonstrated. The optimal conditions of this process (sodium acetate buffer, pH 7.0-7.5; temperature, 40-45 degrees C; and duration of hydrolysis, 3 h) were determined. Depending on the ratio between heparin and the immobilized enzymatic complex, samples with molecular weight varying from 1.7 to 4.7 kDa, were obtained. These complexes inhibited factor Xa in 2.0-3.7 times more effectively than original heparin.

[Antibacterial effects of water-soluble low-molecular-weight chitosans on different microorganisms]. / Antibakterial'naia aktivnost' vodorastvorimykh nizkomolekuliarnykh khitozanov v otnoshenii razlichnykh mikroorganizmov.

Low-molecular-weight chitosans with a viscosity-average molecular weight (Mv) of 5 to 27 kDa and equal degree of deacetylation (DD, 85%) were highly active against Pseudomonas aureofaciens, Enterobacter agglomerans, Bacillus subtilis, and Bifidobacterium bifidum 791, causing death of 80 to 100% of cells. An exception to this tendency was Escherichia coli, for which the rate of cell death, induced by the 5-kDa chitosan, was 38%. The antibacterial effect was manifested as early as 10 min after incubation of 12-kDa chitosan with B. subtilis or E. coli cells. Candida krusei was almost insensitive to the above crab chitosans. However, Candida krusei was highly sensitive to chitosans with Mv 5, 6, 12, 15.7, and 27 kDa: the minimum inhibitory concentration (MIC) varied from 0.06 to 0.005%. Chitosans with M, 5, 12, and 15.7 kDa exerted an antibacterial effect on Staphylococcus aureus. Chitosans with Mv 5, 15.7, and 27 kDa had no effect on Bifidobacterium bifidum ATCC 14893. The antibacterial effect of the 4-kDa chitosan on E. coli and B. bifidum 791 increased with DD in the range 55-85%.

[Hydrolysis of chitosan sulfate by an enzyme complex from Streptomyces kurssanovii]. / Gidroliz sul'fata khitozana fermentnym kompleksom iz Streptomyces kurssanovii.

The possibility of enzymatic hydrolysis of chitosan was shown. The optimum conditions for the process are: sodium acetate buffer pH 6.0, 37 degrees C, 24 h, and the chitosan sulfate-protein volume ratio of 500:1 in the enzyme preparation. During hydrolysis, the intrinsic viscosity of chitosan sulfate solution decreased by a factor of 2.7.

Isolation of thermostable phosphatase from the hyperthermophilic archaeon Thermococcus pacificus by immobilized metal affinity chromatography.

Phosphatase was isolated from cells of the hyperthermophilic marine archaeon Thermococcus pacificus by a procedure including chromatography on Butyl-Fractogel TSK-650 and Ni(2+)-iminodiacetic-agarose. Enzyme activity is maximal at 90 degrees C, and the enzyme half-life time at this temperature is 1 h. The pH optimum of phosphatase activity is 6.0. Electrophoresis under denaturating conditions yielded a subunit molecular weight of 45 kDa. On gel-filtration on Sephacryl S-300 HR three peak corresponding to 295, 85 and 45 kDa were observed, suggesting that the enzyme is a homohexamer.

[Purification of acetylcholinesterase from human erythrocytes using metal-chelate affinity chromatography]. / Ochistka atsetilkholinésterazy iz éritrotsitov cheloveka s pomoshch'iu metallo-khelat affinnoi khromatografii.

Acetylcholinesterase (AChE) was purified on columns with iminodiacetate Agarose charged with Cu2+, Zn2+, and Ni2+. The best results (a 14-fold purification and more than 30% activity yield) were obtained with Zn2+ used as a complex-forming metal. The preparation had a specific activity of approximately 7 U. Its purity was tested by polyacrylamide gel electrophoresis under denaturing conditions.

New approaches to chromatographic purification of bovine dopamine-beta-hydroxylase.

The use of the traditional scheme for the isolation of bovine dopamine-beta-hydroxylase (bDBH) from bovine adrenal medulla resulted in active but not pure bDBH, containing about 50% of admixtures. Immobilized metal chelate affinity chromatography on agarose modified with iminodiacetic acid residues and charged with cobalt ions was applied in the final stage to obtain more than 90% pure and active bDBH. Final purification of bDBH using step elution with 0-0.5 M methyl-D-mannoside in buffer solution from concanavalin A-Sepharose was studied. The determination of bDBH in various samples was performed using size-exclusion chromatography.

[New acetylcholinesterase inhibitors--derivatives of vitamin B6]. / Novye ingibitory atsetilkholinésterazy--proizvodnye vitamina B6.

A new approach to design of reversible inhibitors of acetylcholinesterase (ACHE)--derivatives of natural compounds--has been worked out, as exemplified by vitamins of the B6 group. Analysis of the data obtained revealed main structural elements of the 3-hydroxypyridine molecule related to the inhibitory properties. Pyridoxamine derivatives are of interest in constructing new potent inhibitors of ACHE.

[Isolation of intracellular inhibitors of bacterial RNAses on a column with immobilized Bacillus intermedius RNAse]. / Vydelenie vnutrikletochnykh ingibitorov RNKaz bakterii na kolonke s immobilizovannoi RNKazoi Bacillus intermedius.

Intracellular inhibitors of RNases from Bacillus amyloliquefaciens and Bac. intermedius were isolated using affinity chromatography on covalently immobilized RNase from Bac. intermedius. The inhibitor of RNase from Bac. amyloliquefaciens was isolated from cells of E. coli HB 101 and purified to homogeneity. Proteins with molecular weights of 70, 36 and 20 kD possessing an inhibitory activity were found in the extract obtained from frozen cells of Bac. intermedius.

Two isoforms of Serratia marcescens nuclease. Crystallization and preliminary X-ray investigation of the enzyme.

Two isoforms of an extracellular endonuclease, nucleases Sm1 and Sm2, were purified from culture fluid of Serratia marcescens strain BIO MI by ligand-exchange chromatography on phosphocellulose and DEAE-Toyopearl 650S. The pI-values for nucleases Sm1 and Sm2 were found to be 7.1 and 6.7, respectively. The amino acid analysis and N-terminal amino acid sequencing of the proteins showed a significant degree of homology between the enzymes. The nuclease Sm1 has been crystallized from ammonium sulfate solution by the vapour diffusion technique. The crystals belong to the space group P2(1)2(1)2(1) with unit cell constants a = 69.0, b = 106.7, c = 74.8 A, contain two molecules in an asymmetric unit, packing density Vm = 2.3 A/Da, and diffract to at least 1.5 A resolution. The Pt- and UO2-derivatives of the protein were obtained. Preliminary X-ray investigation of nuclease Sm2 crystals was carried out.

[Isolation of isoforms and crystallization of endonucleases of Serratia marcescens]. / Razdelenie izoform i kristallizatsiia éndonukleazy Serratia marcescens.

Two isoforms of an extracellular endonuclease, nuclease Sm1 and nuclease Sm2, were isolated from the culture filtrate of Serratia marcescens strain B10 M1 by the ligand-exchange chromatography on iminodiacetate-agarose in Cu2(+)-form, and chromatography on phosphocellulose and DEAE-Toyopearl 650S. The pI for nucleases Sm1 and Sm2 were found to be 7.1 and 6.7, respectively. The amino acid analysis and N-terminal amino acid sequencing of the proteins showed a significant degree of homology between the enzymes. The secondary structure of nuclease Sm2 was calculated. Crystals of nuclease Sm2 were obtained with the space group P2(1)2(1)2(1), a 69.0; b 106.7; c 74.8 A.

Ligand-exchange chromatography of nucleases.

The synthesis of chelating sorbents for ligand-exchange chromatography of enzymes is described. An inorganic support "Silochrom" and organic "Spheron", TSK-Gel HW 55 and cellulose were used as initial supports. The chelating sorbents contained iminodiacetic acid and iminodimethylphosphonic acid as stationary ligands. In order to obtain monofunctional sorbents, iminodiacetic acid was added in the form of its dimethyl ester. The concentration of stationary ligands on the sorbents varied from 10 to 100 mumol per ml sorbent. A chelating sorbent (in nickel form) was shown to be effective in the purification of exonuclease A5 from actinomyces. Electrophoretically homogeneous exonuclease A5 was obtained with a 25% yield. A chelating sorbent with iminodiacetic groups (in copper form) was applied to the isolation of endonuclease from Serratia marcescens directly from the culture medium. The capacity of the chelating sorbents for the endonuclease was studied as a function of the stationary ligand concentration. After one stage of purification, more than 70% pure enzyme was obtained with a yield exceeding 80%.

[Affinity chromatography of nucleases]. / Affinnaia khromatografiia nukleaz.

The review concerns isolation and purification of nucleases by affinity chromatography. Different stationary ligands and the methods for their immobilization on supports are described, along with diverse eluents and various procedures for a nuclease detachment from the affinity sorbents. The data on the affinity chromatography application for measuring the dissociation constants of the enzyme complexes with either immobilized or soluble ligands are compiled.