A homogeneous peptide with a mass of 683 Da which inhibits HIV-1 integrase with IC 50 3 × 10 −5 M was separated from aqueous extracts of a marine worm Eunicidae sp. by multistage chromatography purification. The Asp-Leu-Hse-His-Ala-Gln structure was proposed for this peptide according to amino acid analysis, automated amino acid Edman sequences, and TLC with witness homoserine and MS/MS fragmentation. The proposed structure is the first example of a natural peptide containing an amino acid homoserine residue.

The carbohydrate-containing polymer 1,3;1,6-beta-D-glucan was obtained by transformation of laminaran from the alga Laminaria cichorioides with endo-beta-1,3-glucanase from marine mollusks. In electron microscope observations of phosphotungstic acid-stained preparations from Nicotiana tabacum cv. Samsun leaves inoculated with a mixture of Tobacco mosaic virus (TMV) (1 mu g/ml) and glucan (1 mg/ml) or with TMV alone, we found that such preparations contained, along with virus particles of normal diameter (about 18 nm), abnormal (swollen and thinner) particles. The highest number of thin viral particles was found in dips from leaves inoculated with TMV together with glucan. It is suggested that this may be caused by a glucan-mediated increase of TMV particle proteolysis in infected 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-β-D-glucan, fucoidan, and κ/β-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.

The fragmentation of the biologically active 1,3;1,6-β-D-glucan Antivir by endo-1,3-β-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-β-D-glucooligo-and polysaccharides was found to be associated with molecular mass (polymerization degree (n) not less than 14) and with presence of intralinked β-1,6-connected monosaccharide residues. Probably, decrease in molecular mass is compensated by increase in number of intralinked β-1,6-connected monosaccharide residues.

Daucosterol, a glucoside of β-sitosterol, has been isolated from the roots ofAcanthopanax sessiliflorum.

β-1,3-Glucanase (Lu) was isolated from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. A comparative study of some properties of β-1,3-glucanase Lu and β-1,3-glucanases with different action types—endo-β-1,3-glucanase from crystalline style of the marine mollusk Spisula sachalinensis (LIV) and exo-β-1,3-glucanase from the terrestrial snail Eulota maakii (LII)—was performed. It was found that β-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 β-1,3-glucanase from S. intermedius were more similar to those of the endo-β-1,3-glucanase from the marine mollusk (LIV) than exo-β-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 β-1,3-glucanase Lu from sea urchin eggs (which, in contrast to LIV, is not a digestive enzyme).

An α-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 α-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°C and for a week at 20°C. The enzyme can be stored under the sterile conditions for any length of time at 4°C, but it does not withstand freezing. The α-N-acetylgalactosaminidase is resistant to NaCl; for p-nitrophenyl-α-N-acetyl-D-galactosaminide, the Km is 0.38 mM. The molecular mass of the enzyme determined by gel filtration is 84 kD.

The characteristics of carrageenans and agars of red seaweeds from the Russian Pacific coast were investigated. The occurrence of laminarans in brown algae of the Okhotsk Sea and Sea of Japan was determined and their structures were studied. The enzymatic transformations of kappa- and lambda-carrageenans and laminarans were performed and as a result some new biological active polysaccharides were obtained.

Data on the distribution among marine invertebrates of such hydrolyses as 1,3-beta-glucanases, proteinases, and deoxyribonucleases as well as their properties and specificity demonstrate that these enzymes are distinguished from the same type of hydrolyses isolated from other sources. This data are a result of systematic investigations carried out by the Pacific Institute of Bioorganic Chemistry of the Far East Division of the Russian Academy of Sciences (PIBC). These investigation results have expanded the modem views of molecular mechanisms function of this type of hydrolyses as well its possible role in marine life. The identification of new hydrolytic enzymes with unique properties and specificity among the substances isolated from marine invertebrates presents the possibility for new methods of use for biotechnology and medicine.

The action of sulfated polyhydroxysteroids from starfish and brittle stars on the activity of β-1,3-glucanases from various sources has been investigated. All the substances activated the exoglucanase LII, nine out of ten activated Le, and seven out of ten of the sulfates activated the endoglucanase LIII. Other endo-β-1,3-glucanases from marine molluscs were specifically inhibited by the steroid sulfates investigated, and it was shown that the efficiency of inhibition depends on the structural features of the molecules of these inhibitors.

The capacity of certain extracts of marine invertebrates of the Indian and Pacific Oceans for activating β-1,3-glucanases has been detected. It has been shown that an individual compound — swinholide A from the spongeTheonella swinhoei — specifically activates β-1,3-glucanases of bivlve mollusks. In the presence of swinholide A both the hydrolysis of laminarin by β-1,3-glucanase L-IV fromSpisula sachalinensis and the transglycosylation reaction are accelerated.

The effects of natural polyhydroxysteroid sulfates and their analogs on beta-1,3-glucanases with various action mechanisms were investigated. The mode of influence of these substances on the activity of exo- and endoenzymes differs. For endoenzymes, sulfated polyhydroxysteroids are effective inhibitors. Exo-enzymes are inhibited only at significant concentrations of the sulfated polyhydroxysteroids: low concentrations produce appreciable activation. The efficiency of endo-beta-1,3-glucanase inhibition depends on the structural characteristics of the investigated compounds and on the nature of the glucanases. The interaction of chalistanol sulfate with the endo-beta-1,3-glucanases LIV and LO from marine mollusks was investigated by methods of intrinsic fluorescence, UV absorption spectrophotometry, and CD spectroscopy. The binding of chalistanol sulfate to glucanases was found to induce a conformational translation in the molecules of these enzymes. The binding constants K(a) were determined: 2.9.10(4) M-1 for LIV and 6.5.10(4) M-1 for LO; the stoichiometric coefficients were found to be equal to 2.7 for LIV and 1.8 for LO.

The distribution of the products of the enzymatic transformation of various substrates under the action of the endo-β-(1 → 3)-glucanase L IV from the molluscSpisula sachalinensis has been investigated and the possibility has been shown of the simultaneous recording, with the aid of EDIAP mass spectrometry, of the products of hydrolysis and of transglycosylation. It has been shown that methyl gentiobioside and methyl cellobioside are acceptors in the transglycosylation reaction, as a result of which glucooligosaccharides with a mixed type of glycosidic bonds are formed. The ratio of mono-, di-, tri-, and tetrasaccharides labeled at the ends with ethylene glycol formed in the enzymatic hydroysis of laminarin previously subjected to Smith degradation has been determined.