Correlation between influence of polysaccharides on hydrolase activity and their antiviral effect in tobacco leaves.

The activities of hydrolases (acid phosphatase, RNase, and proteases) in healthy and tobacco mosaic virus-infected leaves of Nicotiana tabacum L. var. Samsun, both untreated and treated with polysaccharides (PS) (1,3;1,6-ß-D-glucan, fucoidan, and κ/ß-carrageenan), were determined. The PS lead to substantial increase in the hydrolase level. The percentage of viral particles undergoing destructive change also increases in leaves treated with PS 24 h before infection. We suppose that the PS-mediated hydrolase activation promotes intracellular destruction of the viral particles and, thus, comprises one of the PS-induced protective mechanisms limiting intracellular viral accumulation.

Enzymatic transformation of biologically active 1,3;1,6-beta-D-glucan. Structure and activity of resulting fragments.

The fragmentation of the biologically active 1,3;1,6-beta-D-glucan Antivir by endo-1,3-beta-D-glucanase LIV from crystalline styles of the marine mollusk Spisula sachalinensis was carried out. It was found that low molecular mass oligomers possessing a stabilizing effect on membranes and anti-viral activity against tobacco mosaic virus appeared in the process of enzymatic hydrolysis of Antivir. Biological activity of 1,3;1,6-beta-D-glucooligo- and polysaccharides was found to be associated with molecular mass (polymerization degree (n) not less than 14) and with presence of intralinked beta-1,6-connected monosaccharide residues. Probably, decrease in molecular mass is compensated by increase in number of intralinked beta-1,6-connected monosaccharide residues.

Beta-1,3-glucanase from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. Comparison with beta-1,3-glucanases of marine and terrestrial mollusks.

beta-1,3-Glucanase (Lu) was isolated from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. A comparative study of some properties of beta-1,3-glucanase Lu and beta-1,3-glucanases with different action types--endo-beta-1,3-glucanase from crystalline style of the marine mollusk Spisula sachalinensis (LIV) and exo-beta-1,3-glucanase from the terrestrial snail Eulota maakii (LII)--was performed. It was found that beta-1,3-glucanase Lu hydrolyzes laminaran with a high yield of glucose in the reaction products. The enzyme hydrolyzes substrates with retention of the glycosidic bond configuration, is able to cleave modified substrates, and exhibits transglycosylating activity. All properties of beta-1,3-glucanase from S. intermedius were more similar to those of the endo-beta-1,3-glucanase from the marine mollusk (LIV) than exo-beta-1,3-glucanase LII from the terrestrial snail. The differences in the effect of LIV and Lu on laminaran are probably related to the functions of beta-1,3-glucanase Lu from sea urchin eggs (which, in contrast to LIV, is not a digestive enzyme).

Alpha-N-acetylgalactosaminidase from marine bacterium Arenibacter latericius KMM 426T removing blood type specificity of A-erythrocytes.

An alpha-N-acetylgalactosaminidase IV able to remove blood type specificity of human A(II)-erythrocytes and not effecting B(III)-erythrocytes was isolated from the marine bacterium Arenibacter latericius KMM 426T. The alpha-N-acetylgalactosaminidase IV preparation exhibits high activity during inhibition of hemagglutination with blood group substance A containing determinants analogous to A-erythrocytes. The enzyme has a pH optimum from 7.0 to 8.0 and completely retains its activity during 30-min heating at 50 degrees C and for a week at 20 degrees C. The enzyme can be stored under the sterile conditions for any length of time at 4 degrees C, but it does not withstand freezing. The alpha-N-acetylgalactosaminidase is resistant to NaCl; for p-nitrophenyl-alpha-N-acetyl-D-galactosaminide, the Km is 0.38 mM. The molecular mass of the enzyme determined by gel filtration is 84 kD.

Inhibition of complement activation by water-soluble polysaccharides of some far-eastern brown seaweeds.

Fucoidans and laminarans from Laminaria cichorioides, Laminaria japonica, Fucus evanescens, laminaran from Laminaria gurjanovae, other beta-D-glucans (translam, pustulan and zymosan) and lambda-carrageenan from Chondrus armatus were used to study the effect of water-soluble polysaccharides from seaweeds on the alternative pathway of complement (APC). beta-D-Glucans and fucoidans under study differed appreciably from each other by structural characteristics, and also by degree of purification. beta-D-glucans, on ability to bind complement, ranked in a line according to a degree of their purification. Highly purified beta-D-glucans under study did not reveal an ability to bind complement. The fucoidans were divided conventionally into three groups according to their action on APC. Highly sulfated alpha-L-fucan from L. cichorioides with the greatest activity toward APC and caused 50% inhibition of reaction of activation (RA) of APC in a concentration of 0.5-0.7 mg/ml. Opposite 50% of inhibition of lysis of erythrocytes by sulfated heterogeneous fucoidan from L. japonica was achieved with 20 mg/ml. All other fucoidans and lambda-carrageenan have activity at 6-10 mg/ml concentration. Decreasing the sulfate content from 36% up to 9% in sample fucoidans under study was not reflected practically in the 50% inhibition concentration. Apparently, the degree of sulfating of fucoidans did not influence their action on APC. But the positive influence of fucose in structure of polysaccharide was obvious.

Alpha-galactosidase of the marine bacterium Pseudoalteromonas sp. KMM 701.

An alpha-galactosidase that inactivates the group specificity of B erythrocytes (group III) of human blood and does not affect A erythrocytes (group II) was isolated from the marine bacterium Pseudoalteromonas sp. KMM 701. The enzyme preparation did not contain lectin, hemolytic, sialidase, endoglycanase, or glycosidase activities. The enzyme is stable at 20 degreesC for 24 h, has pH optimum for catalysis within the range of 6.7-7.7, and is stable to high concentrations of NaCl. It is 4-fold more efficient than the alpha-galactosidase from green coffee beans. At pH 7.0 the Km for p-nitrophenyl-alpha-D-galactopyranoside is 0.29 mM. The molecular weight of the enzyme determined by gel-filtration is 195 +/- 5 kD. The alpha-galactosidase is denatured by urea and guanidine hydrochloride. Its activity does not depend on the presence of metal ions. It contains a sulfhydryl group essential for its catalytic activity.

Investigation of the conformational properties of a beta-(1-->3) branched beta-(1-->6) heptasaccharide elicitor and its analogues by internal coordinate stochastic dynamics.

A series of beta-(1-->3) branched beta-(1-->6) oligosaccharides that are known to take part in switching immune reactions in plants was studied by a molecular dynamics approach. A novel technique was applied which recently proved to be very efficient in polypeptide simulations. Molecular dynamics is simulated in internal rather than Cartesian coordinates with dramatically reduced numbers of degrees of freedom and a time step ten-fold larger than usual values. Comparison and classification of most populated conformational states revealed a few conformational motifs that are frequently adopted by highly active oligosaccharides and are not populated in an inactive analogue. As a result a putative biologically active conformation of the oligosaccharides is proposed.

The search for effectors of beta-D-glucanases among marine invertebrates.

1. The search for inhibitors and activators of endo-1----3-(EC 3.2.1.6) and 1----6-beta-D-glucanases (EC3.2.1) from marine mollusks in aqueous and ethanol extracts of various types of marine invertebrates, such as sponges, ascidians, coelenterates, echinoderms, dwelling in north seas (the sea of Okhotsk and the sea of Japan) and torrid seas (western coast of Australia) has been made. 2. A comparative analysis has shown that the overwhelming majority of extracts of animals, dwelling near Australia, inhibit beta-D-glucanases. 3. On the contrary, the extracts of animals dwelling in the sea of Okhotsk possess the activating effect, except for sponges of genera Haliclona whose sample extracts display a significant activating effect independently of their place of abode.

A study of the reaction catalysed by alginate lyase VI from the sea mollusc, Littorina sp.

The molecular weight of polymeric alginic acid digested by alginate lyase (poly(1,4-beta-D-mannuronide) lyase, EC 4.2.2.3) was determined at various stages of the lysis. Low molecular weigh fragments were detected only after 60-100% lysis. Some high molecular weight fragments remained intact even after addition of a fresh aliquot of enzyme to the digest. The enzyme showed maximal activity at pH 5.6 in 0.05 M salt. Enzyme activity was stimulated by addition of 7.5 mM CaCl2 and 0.2 M NaCl, when the pH optimum was between 8 and 8.5. Only mannuronic acid was detected at the reducing end of fragments after exhausive enzymolysis, reduction and hydrolysis. On studying the reaction products by NMR, a double-bound signal (sigma = 5.98 ppm) was observed. A considerable decrease in intensity of the D-mannuronic acid residue signal was detected after hydrolysis of alginate lyase VI on poly-(ManUA-GulUA), but not poly(GulUA). The results suggest that alginate lyase VI may be an endoalginate lyase that splits glycoside bonds only between two mannuronic acid residues.