Functional properties of individual sub-domains of the fibrin(ogen) αC-domains.

Background: Fibrinogen is a large polyfunctional plasma protein consisting of a number of structural and functional domains. Among them, two αC-domains, each formed by the amino acid residues Аα392-610, are involved in fibrin polymerization, activation of fibrinolysis, platelet aggregation, and interaction with different cell types. Previous study revealed that each fibrinogen αC-domain consists of the N-terminal and C-terminal sub-domains. The major objections of the present study were to test functional role of these sub-domains in the above mentioned processes. Methods: To achieve these objections, we used specific proteases to prepare two truncated forms of fibrinogen, fibrinogen desAα505-610 and fibrinogen desAα414-610, missing their N-terminal and both N- and C-terminal sub-domains, respectively. Results: Our study with these truncated forms using turbidity measurements and electron microscopy revealed that the N- and C-terminal subdomains both contribute to protofibril formation and their lateral aggregation into fibers during fibrin polymerization process. These two sub-domains also contributed to platelet aggregation with the N-terminal sub-domains playing a more significant role in this process. At the same time, the C-terminal sub-domains make the major contribution to the plasminogen activation process. Further, our experiments revealed that the C-terminal sub-domains are involved in endothelial cell viability and migration of cancer cells. Conclusions: Thus, the results obtained establish the functional role of individual sub-domains of the αC-domains in fibrin polymerization, activation of fibrinolytic system, platelet aggregation, and cellular interactions. General significance: The present study expands our understanding of the functional role of individual fibrinogen domains and their specific portions in various fibrin(ogen)-dependent processes.

Characterization of the lipopolysaccharide of Escherichia coli 126.

The lipopolysaccharide (LPS) of Escherichia coli 126 was isolated and studied. The lipid A fatty acid composition of the investigated LPS was similar to that of other members of the family Enterobacteriaceae. The E. coli 126 LPS was more toxic than the LPSs of previously studied E. coli strains and of other members of the Enterobacteriaceae (Budvicia aquatica and Pragia fontium), and was less pyrogenic than pyrogenal. SDS-PAG electrophoresis showed a bimodal distribution typical of S-form LPSs. The LPS of E. coli 126 decreased the adhesive index indicating a possible competition between LPS molecules of E. coli 126 and adhesins of E. coli F-50 on rabbit erythrocytes. The LPS of E. coli 126 in a homologous system showed antigenic activity in the reactions of double immunodiffusion in agar by Ouchterlony. No serological cross-reaction of the LPS of other E. coli strains, as well as of that of the B. aquatica type strain, with the antiserum to E. coli 126 was observed. The structural components of the lipopolysaccharide obtained by mild acid hydrolysis were lipid A, the core oligosaccharide, and the O-specific polysaccharide. Based on the data of monosaccharide analysis and 1H and 13C NMR spectroscopy it was found that the O-specific polysaccharide had the structure characteristic of the representatives of E. coli serogroup O15.

Isolation and purification of Bacillus thuringiensis var. israelensis IМV В-7465 peptidase with specificity toward elastin and collagen.

Peptidase of Bacillus thuringiensis var. israelensis IМV В-7465 was isolated from culture supernatant using consecutive fractionations by an ammonium sulphate (60% saturation), ion-exchange chromatography and gel-filtration on the TSK-gels Toyoperl HW-55 and DEAE 650(M). Specific elastase (442 U∙mg of protein-1) and collagenase (212.7 U∙mg of protein-1) activities of the purified enzyme preparation were 8.0- and 6.1-fold, respectively higher than ones of the culture supernatant. Peptidase yields were 33.5% for elastase activity and 30.1% for collagenase activity. It was established that the enzyme is serine metal-dependent alkaline peptidase with Mr about 37 kDa. Maximal hydrolysis of elastin and collagen occurs at the optimum pH 8.0 and t° ­ 40 and 50 °Ð¡, respectively. The purified preparation has high stability at pH in the range of 7.0 to 10.0 and 40-50 °Ð¡.

[The Effect of Metal Ions and Chemical Reagents on the Activity of Achromobacter sp. 7a α-Amylase].

The effect of cations and anions on the activity of Achromobacter sp. 7a α-amylase was studied. It is shown that tested enzyme is stable to most of anions, however sensitive to a number of cations. The most significant inhibitory effects on the activity of Achromobacter sp. 7a α-amylase exerted Hg2+, Al3+, Fe3+, Cu2+ and Ag+ ions. Decline of activity Achromobacter sp. 7a α-amylase in the presence of EDTA and EGTA indicated on the presence within its structure of metal ions. An important role in the functioning of this enzyme play a carboxyl and sulfhydryl groups as evidenced by its inhibition of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide and p-chloromercuribenzoate respectively. α-Amylase Achromobacter sp. 7a does not contain histidine imidazole group in the active center, unlike most studied glycosidases. The tested enzyme showed high stability in the presence of Tween-20, urea, peroxide of hydrogen, making it competitive with previously described α-amylases.

[Screening of the Producents of α-L-Rhamnosidases Amongst Representatives of Penicillium].

The aim of this work was to study α-L-rhamnosidase [КФ 3.2.1.40] - enzyme, which hydrolyse the terminal non-reduced α-1,2-, α-1,4- and α-1,6-linked L-rhamnose. As a result of screening conducted among 30 strains of micromycetes ability to synthesize α-L-rhamnosidase revealed in Penicillium tardum 60, 39, 2929, 2962, 2963, 2964, 2965, 2966, P. rugulosum 2778, 1652, 2766, P. restrictum 425, 2756, P. aculeatum 202, 217, 329, 2973, 2974, 2975, 2976, 2977, 2979 activity, which ranged from 0.07 to 0.53 OD/mg protein. The most active is brought out P. amleatum 202. From culture supernatant of this micromycete by fractionation with ammonium sulfate (90 % saturation) complex enzyme preparation was obtained and its physico-chemical properties were studied. It was shown that enzyme has pH optimum 3.0, thermooptimum - 60 °C and displayed stability in pH values from 2.0 to 4.0 during 90 min. At pH 5.0 the activity of complex enzyme preparation insignificantly decreased and appears to be up 40 % from initial one. At optimal pH value 3.0 and temperature 15 °C α-L-rhamnosidase tested was stable during 3 days. In addition to α-L-rhamnosidase enzyme preparation of P. arnleatum 202 exerted also ß-D-glucosidase activity.

[Screening of α-L-Rhamnosidases and Peptidases among Actinobacterium and Bacilli].

Purpose: To carry out screening of peptidases and α-L-rhamnosidases producers among actinobacterium and bacilli. Methods: The biochemical methods of α-L-rhamnosidase, elastase, caseinolytic, fibrinolytic and collagenase activity determination have been used. Results: Among 31 strains of actinobacterium and 24 strains of bacilli it was not exhibited any enzyme with elactolytic activity, while a number of actinobacterium strains displayed high collagenase activity. As to bacilli, elastolytic activity was observed only in five strains, however its level is not an interest for future investigations. Bacillus subtilis 121 and 108 exerted enough high activity (0.100 and 0.092 U/mg of protein respectively). Conclusion: The most effective producer of collagenase and α-L-rhamnosidase is actinobacterium strain 6/5 isolated from nettle zhisosphere,while peptidase with fibrinolytic activity - B. subtilis 121 and 108. We believe these strains may be perspective for further researches.

[Functional and Biological Activity of Pantoea agglomerans Lipopolysaccharides].

The lipopolysaccharides (LPS) of the seven strains of Pantoea agglomerans were isolated and chemically identified. It was established that the investigated strains characterized by different relative output of LPS from 5.2 to 14.0 % by dry weight of bacteria. LPS were characterized quite high content of carbohydrates - from 22 to 54 % 2-keto-3-deoxyoctonic acid (KDO) - from 0.39 to 2.22 % and heptose - from 3.3 to 14.00 %. Fatty acids, containing in the chain of 12 to 16 carbon atoms were identified. Lipids A of all tested LPS were characterized by predominant 3-OH-C14:0 acid from 31.7 to 39.3 % depending on the strain. Since all the studied strains of P. agglomerans were sensitive to polymyxin B, it can be concluded that the LPS do not contain in the structure of lipid A, such a substitute as 4-amino-4-deoxy-L-arabinose. One of the ways of changes in the functional and biological properties of LPS is the chemical modification. As modifiers were used complexes of germanium and tin. In the study of serological activity and toxicity of modified LPS it was found that some of them lost both serological and toxic activity. It was revealed that all investigated P. agglomerans LPS decreased the median adhesion and the index of the adhesiveness. The higher concentration of P. agglomerans LPS in the reaction mixture, the less interactions between surface structures of red blood cells and E. coli cells.

[Screening of Mannane-Degrading Enzymes Producers].

The aim of research was to investigate the prevalence of complex mannan-degrading enzymes among museum and freshly isolated cultures of micromycetes, actinobacteria and bacteria. It has been shown that the producers of ß-mannanases mostly extracted from sources that are rich in plant residues. We detected strains of Penicillium aculeatum, P. tardum and P. rugulosum which produce a complex of ß-mannanases and α-galactosidase activity. Also, strains of thermophilic micromycetes species Corynascus sepedonium, Scytalidium thermophilum and Rhizomucor tauricus with high mannanase activity in culture liquid were detected (10 ­ 130 U/ml). Two strains of P. aculeatum and P. tardum showed ß-mannanase, ß-mannosidase and α-galactosidase activity. Actinobacteria was shown high potential, 70 % of all tested strains showed mannanase activity; their activity ranged from 2 to 55 U/ml. Among the most active bacterial cultures were representatives of soil microflora: Bacillus circulans, B. subtilis, B. mesentericus, where as among the strains isolated from sea water the active mannanases producers were not found.

Structure, Function and Biological Activity of Lipopolysaccharide Lipid A.

Bacterial lipopolysaccharides (LPS) are the major outer surface membrane components present in almost all gram-negative bacteria. It consists of poly- or oligosaccharide region that is anchored in the outer membrane by a specific lipid moiety termed lipid A. Recent studies have shown that it is only the lipid A of LPS that has the function of endotoxin. Despite its general structural conservation, lipid A also has considerable structural microheterogeneity which can vary depending on diverse factors including bacterial adaptation to changing environment and external stimuli, incomplete biosynthesis, and breakdown products and/or chemical modifications. Therefore it is more appropriate to consider lipid A as a family of structurally related molecular species with different acylation and phosphorylation patterns rather than as an individual, homogeneous molecule. The studies of structure-function relationship of lipid A, which has the typical structure of E. coli type lipid A backbone, demonstrated that activities differed depending on: 1) the number of phosphoryl and acyl residues, 2) the substituted site of phosphoryl and acyl residues, 3) the chain length of acyl residues, 4) lipid A conformation. Current investigations showed that lipid A and also the integral outer membrane proteins responsible for the final stage of LPS transport are the pinpoints in solving the problem of bacterial drug resistance. The identification of inhibitors that specifically target LPS transport in vitro and more importantly in vivo have a significant potential for the development of novel drugs against multi-drug resistant pathogenic bacteria.

[PURIFICATION AND PHYSICO-CHEMICAL PROPERTIES OF PENICILLIUM TARDUM a-L- RHAMNOSIDASE].

By ammonium sulfate fractionation and chromatography on TSK-gels Toyopearl HW- 60 and Fractogel TSK DEAE-650-s from supernantant of cultural liquid of Penicillium tardum 1MB F-100074 was isolated and purified in 23 times preparation of enzyme with α-L-rhamnosidase activity with yield of 3.12 %. The specific activity was 27.7 U/mg of protein, molecular mass 67 kDa, thermo- and pH optimum - 60 °C and pH 5.0 respec- tively. It was shown high pH- and thermostability of purified P tardum a-L-rhamnosidase which may be dued to presence of 12 % of carbohydrate component.

[Physico-Chemical Properties of Achromobacter SP. α-Amylase].

From Achromobacter sp. 7a, that was isolated with Black sea, aquatoria of island Zmi- inyi, was isolated enzyme with α-amylase activity, that able also to split the synthetic substrates: p-nitrophenyl-α-D-glucopyranoside and p-nitrophenyl-α, -ß-D-xylopyranoside. Methods of isolation and purification of enzyme were selected which included: ammonium sulfate precipitation and affinity sorption on starch, that improved enzyme activity in 7 times in comparison with activity in the supernatant of cultural liquid. a-Amylase showed maximal activity at pH 7.0 and 11.0 and to the temperature 50 °C. Enzyme remained fully stable during 24 hours in the range of pH from 7.0 to 12.0, during 3 hours at a temperature 37 °C and 50 °C at pHopt 7.0, and also 87.5 % and 75 % of initial activity saved during 3 h of incubation at a temperature 37 °C and 50 °C at pHopt 11.0 respectively. It is shown that addition of antihunt agents (ions of calcium, chloride of natrium) did not protect an enzyme from thermoinactivation (60 °C, 70 °C).

STABILITY OF NATIVE AND MODIFIED α-GALACTOSIDASE OF Cladosporium cladosporioides.

By modifying carbohydrate component of glycoproteins it is possible to elucidate its role in manifestation of structural and functional properties of the enzyme. The comparison of activity and stability of the native and modified by oxidation with sodium periodate α-galactosidase of Cladosporium cladosporioides was carried out. To determine α-galactosidase activity the authors used n-nitrophenyl synthetic substrate, as well as melibiose; raffinose and stachyose. Modification of the carbohydrate component had a significant effect on catalytic properties of the enzyme. Both the reduction of V and enzyme affinity for natural and synthetic substrates were observed The native enzyme retained more than 50% ofthe maximum activity in the range of 20-60 °C, while for the modified enzyme under the same conditions that temperature range was 30-50 °C. The modified α-galactosidase demonstrated a higher thermal stability under neutral pH conditions. The residual activity of the modified α-galactosidase was about 30% when treated with 70% (v/v) methanol, ethanol and propanol. About 50% of initial activity was observed when 40% ethanol and propanol, and 50% methanol were used. It was shown that the modification of C. cladosporioides α-galactosidase by sodium periodate is accompanied by a significant decrease in enzyme activity and stability, probably caused by topological changes in the tertiary and quaternary structure of the protein molecule.

THERMAL STABILITY OF Cryptococcus albidus α-L-RHAMNOSIDASE.

Yeast as well as micromycetes α-L-rhamnosidases, currently, are the most promising group of enzymes. Improving of the thermal stability of the enzyme preparation are especially important studies. Increase in stability and efficiency of substrate hydrolysis by α-L-rhamnosidase will improve the production technology of juices and wines. The aim of our study was to investigate the rate of naringin hydrolysis by α-L-rhamnosidase from Cryptococcus albidus, and also some aspects of the thermal denaturation and stabilization of this enzyme. We investigated two forms of α-L-rhamnosidase from C. albidus, which were obtained by cultivation of the producer on two carbon sources--naringin and rhamnose. A comparative study of properties and the process of thermal inactivation of α-L-rhamnosidases showed that the inducer of synthesis had no effect on the efficiency of naringin hydrolysis by the enzyme, but modified thermal stability of the protein molecule. Hydrophobic interactions and the cysteine residues are involved in maintaining of active conformation of the α-L-rhamnosidase molecule. Yeast α-L-rhamnosidase is also stabilized by 0.5% bovine serum albumin and 0.25% glutaraldehyde.

[OPTIMIZATION OF CULTIVATION CONDITIONS OF PENICILLIUM TARDUM--THE α-L- RHAMNOSIDASE PRODUCER].

The influence of some technological cultivation parameters of Penicillium tardum to synthesize of the extracellular α.-L-rhamnosidase were studied. It was shown that rhamnose (0.8%), yeasts autolysate (0.2%), temperature of the cultivation 25 degrees C, pH 5.0 are necessary for maximal α-L-rhamnosidase production. The enzyme reaches the maximal activity level in 96 hours with sulphitic number equal 0.44. At cultivation of P. tardum in the picked up conditions the α-L-rhamnosidase synthesis has raised in 4 times.

[THE EFFECT OF METAL IONES AND SPECIFIC CHEMICAL REAGENTS ON THE ACTIVITY OF ASPERGILLUS FLAVUS VAR. ORYZAE AND BACILLUS SUBTILIS α-AMYLASES].

The effect of cations and anions on the activity of Aspergillus flavus var. oryzae and Bacillus subtilis α-amylases showed that the tested enzymes are sensitive to most of cations and resistant to anions. The most significant inhibitory effects on the activity of A. flavus var. oryzae α-amylase have been demonstrated by Al3+ and Fe3+ ions, while on the activity of B. subtilis α-amylase - Hg2+, Cu2+ and Fe3+ ions. Inactivation of A. flavus var. oryzae and B. subtilis α-amylases in the presence of EGTA is indicated on the presence within their structure of metal ions. An important role in the enzymatic catalysis of both enzymes play carboxyl groups as evidenced by their inhibition of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide. Inhibition of B. subtilis α-amylase by p-chloromercuribenzoate, N-ethylmaleimide and sodium sulfite is indicated on the probable involvement of the sulfhydryl groups in the functioning of the enzyme. Unlike most studied glycosidases the tested enzymes do not contain histidine imidazole group in the active center.

[ABILITY OF MICROORGANISMS FROM DIFFERENT ECOLOGICAL NICHES TO HYDROLYZE THE INSOLUBLE PROTEINS].

Screening of protease producers with specificity to insoluble and hard soluble protein substrates of animal origin (collagen, fibrin, elastin and keratin) was carried out. It was studied the bacterial cultures (24 strains) isolated from water and periphyton of enclosures with dolphins, and also from exhalations, oral cavity and skin of dolphins. Some bacterial strains isolated from water and periphyton of enclosures hydrolyzed collagen (5-23 U/ml) and elastin (20-32 U/ml). Thus all tested cultures did not possess the property of extracellular keratinases synthesis. The streptomycetes (48 strains) were isolated from the soil of Black Sea coastal strip near Odessa and Saky, from parkland and the shores of freshwater lake in Saky and from the soil of Atlantic Ocean coastal strip near Albufena (Portugal). Several streptomycetes have been found to appeare the perspective producers of extracellular keratinase and collagenase. The strains isolated from the soil of the coastal strip area both sea and freshwater lake in Saky possessed the highest activity (up to 5 U/mg).

[Characterization of the Lipopolysaccharides of Pseudomonas chlororaphis].

Lipopolysaccharides (LPS) from two strains ot Pseudomonas chlororaphis subsp. aureofaciens,UCM B-111 and UCM B-306, were isolated and characterized. The LPS preparations exhibited low toxicity, high pyrogenicity and high antiviral activity. Mild acid hydrolysis was used to obtain the O-specific polysaccharides. Their structures were established by monosaccharide analysis and determination of absolute configurations, as well as by 1D and 2D NMR spectroscopy. The O-polysaccharides were shown to contain the linear tri- or tetrasaccharide repeating units. Both O-polysaccharides were structurally heterogeneous: P. chlororaphis subsp. aureofaciens UCM B-111--> 4)-αD-GalpNAc6Ac-(1 --> 3)-ß-D-QuipNAc-(1 --> 6)-αD-GlcpNAc-(l --> ßD-GlcpNAc-(l --> 3)] GalNAc -60%; degree of the non-stoichiometric 6-O-acetylation of GalNAc -60%; P. chlorophis subsp. aureofaciens UCM B-306 --> 3)-α-D-Rhap-(1 --> 4)-α-D-GalpNAcAN-(1 --> 3)-αD-QuipNAc4NAc-(1 -->, where GalNAcAN is 2-acetamido-2-deoxy-D-galacturonamide, the degree of non-stoichiometric amidation of the GalNAcA residue -60%.

[SEROLOGICAL PROPERTIES AND BIOLOGICAL ACTIVITY OF PANTOEA AGGLOMERANS LIPOPOLYSACCHARIDES].

The serological and phytotoxic properties of lipopolysaccharide (LPS) of plant pathogens--Pantoea agglomerans were studied. It is known that the thin variations in the structure of the O-specific polysaccharides determining serological specificity of gram- negative bacteria and used as a molecular basis of serological classification schemes. For P. agglomerans still does not exist a classification scheme based on serology specificity of their LPS. The results of cross serological tests demonstrate immunochemical heterogeneity of species P agglomerans. Only three strains of the 8488, 8490 and 7969 according to the agglutination of O-antigens and direct hemagglutination and inhibition direct hemagglutination can be attributed to a single serogroup. Other strains--each separate group, although some have a relationship. Compared with control plants under the influence of seed treatment of LPS in plants may be reduced, and in some cases increased root length, height and weight sprout, depending on the strain from which the selected LPS. Dive seedlings of tomatoes in the solutions of the studied preparations FSC caused the loss, and after some time, restore turgor.

[Component composition of Cryptococcus albidus and Eupenicillium erubescens alpha-L-rhamnosidases].

The component composition of Cryptococcus albidus and Eupenicillium erubescers alpha-L-rhamnosidases have studied. It was shown that enzymes have a monomeric structure. Enzyme preparations of C. albidus and E. erubescens have similar qualitative but differ in quantitative amino acid composition. alpha-L-rhamnosidase of C. albidus characterised by high amount of histidine, proline, cysteine, methionine in compared with alpha-L-rhamnosidase of E. erubescens. alpha-L-Rhamnosidase of E. erubescens, in contrast to the alpha-L-rhamnnosidase of C. albidus, contained higher levels of lysine, arginine, threonine, alanine, isoleucine, leucine, tyrosine, phenylalanine. It is shown that purified preparations of alpha-L-rhamnosidase C. albidus and E. erubescens contained 5 and 1% carbohydrates respectively. Enzyme preparations differ in quantitative monosaccharide composition, which represented by rhanmose, xylose, mannose, galactose and glucose. Furthermore, alpha-L-rhannosidase C. albidus contained fuicose, whereas alpha-L-rhamnosidase E. erubescens--ribose and arabinose. A significant percentage of hydrophobic amino acids, which is 31 and 34% of the total content, and the presence of the carbohydrate component are essential in stabilization of enzymes molecule.

[Characterization of the lipopolysaccharide from Budvicia aquatica DLR 20186].

A lipopolysaccharide (LPS) from Budvicia aquatica DRL 20186 was isolated, studied, and chemically identified. It was shown to be lowly toxic, but highly pyrogenic. Its fatty acid composition was similar to that of the LPS from other Enterobacteriaceae, with predominance of tetradecanoic (32.7%) and 3-hydroxytetradecanoic acids (23.8%). Hexadecenoic (20.4%), hexadecanoic (11.8%), and dodecanoic acids (8.4%) were also revealed. Double immunodiffusion in agar by the Ouchterlony method revealed antigenic activity of the B. aquatica DLR 20186 LPS in a homologous system. In cross reactions, however, it did not interact with the antisera to other @B. aquatica@ strains.