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From the highly purified but lowly active recombinant protein Destabilas-Lysozyme (Dest-Lys) by use cation-exchange column TSK CM 3-SW chromatography, it was separated non-active fraction IV, contained 90% of protein. Fractions I, II and III, represented proteins with lysozyme and isopeptidase activities. Their lysozyme activity correlates with the activity of natural Des-Lys. The ratio of the activities in fractions I - III is such, that maximal lysozyme activity is concentrated in fraction III, isopeptidase - in fraction I. It is discussed the possibility of Dest-Lys different functions regulation is depended on the formation of protein complex forms.

Based on three-dimensional model of the bifunctional enzyme Destabilase-Lysozyme (mlDL-Ds2) in complex with trimer of N-acetylglucosoamine (NAG)3 the functional role of the stereochemically based group of amino acids (Glu14, Asp26, Ser 29, Ser31, Lys38, His92), in manifestation of glycosidase and isopeptidase activities has been elucidated. By method of site-directed mutagenesis it has been shown that mlDL glycosidase active site includes catalytic Glu14 and Asp26, and isopeptidase site functions as Ser/Lys dyad presented by catalytic residues Lys38 and Ser29. Thus, among the invertebrate lysozymes mlDL presents first example of the bifunctional enzyme with identified position of the isopeptidase active site and localization of the corresponding catalytic residues.

Salivary gland secretions of three species of the medicinal leech differ in the level of lysozyme peptidoglycan-lysing activity. Using the synthetic fluorogenic substrate, 4-methyl-umbelliferyl tetra N-acetyl-beta-chitotetraosid, the glycosidase activity (as one of peptidoglycan-lysing activities) of salivary gland secretion of three species of the medicinal leech was quantitatively evaluated in comparison with egg lysozyme. It is supposed, that lysozyme activity of the leech secretions is determined not only by 5 isoforms of destabilase-lysozyme, but by some other enzymes which can utilize this substrate. These may be lysozymes other than i- (invertebrate) lysozymes (such as destabilase-lysozyme, or related enzymes).

Contents of J-peptide of secreted human polymeric immunoglobulins may vary considerably with different pathologies, reflecting the state of the adaptive immune system. In this work assessed the content of J-peptide in various tissues of healthy people to use as a baseline for studies related to the change in the content of J-peptide in pathologies.

Preparation and purification of a recombinant protein are described along with characteristics of its specific (for ε-(γ-Glu)-Lys and D-dimer substrates) and nonspecific (for L-γ-Glu-pNA) isopeptidase activities; the absence of peptidase function for α-(α-Glu)-Lys substrate is noted. It is shown that the protein exhibits muramidase (cell walls of Micrococcus lysodeikticus) and specific glycosidase activities. The latter was determined towards the fluorogenic substrate 4-methylumbelliferyl-tetra-N-acetyl-β-chitotetraoxide. Antimicrobial activity of recombinant destabilase-lysozyme protein (recDest-Lys) and its 11-membered amphipathic peptide was revealed towards cells of the strict anaerobic Archaean Methanosarcina barkeri, whose cell walls contain no murein. Possible mechanisms of the effect of recDest-Lys on these cells are discussed.

The relative location of proteins and lipids in particles of medicinal leech salivary gland secretion (SGS) is revealed for the first time. Their sizes and morphology are described. Using scanning electron microscopy and transmission electron microscopy, it was determined that SGS consists of particles of different sizes and form. This picture is supported by confocal laser scanning microscopy of SGS preparations treated with fluorescein isothiocyanate. After incubation with nonionic detergents (Brij 35 and Tween 20), transmission electron microscopy revealed the dissociation of fragments composing protein-lipid particles (PLP), and in this case an increase in free protein concentration determined by a modification of the Lowry method was observed. Perylene probing of lipids in SGS preparations showed that they are concentrated mainly inside PLP and are almost absent on the surface. Cholesterol was detected during SGS probing using the cholesteryl-Bodipy (hydrophobic fluorescent analog of cholesterol) on surface sections during confocal analysis of electron microphotographs of SGS. This analysis detected PLP structures in SGS resembling caveoles full of cholesterol. SGS, preliminary frozen at -70 degrees C, transformed into a multitude of similar small particles visualized by transmission electron microscopy, whose fixed distribution resembled water crystal structure.

Tumor-specific promoters are predominantly active and ensure expression of the gene under control exclusively in cancer cells. However, a low activity of the promoters is an essential disadvantage for their therapy usage. To achieve a higher expression level of the therapeutic gene, herpes simplex virus thymidine kinase (HSV-tk), the Tat-TAR-system being utilized by HIV-1 for increasing own gene expression was developed. A potentiating activity of tat gene under control of two different cancer-specific gene (human survivin gene and human telomerase reverse transcriptase) promoters for increasing of the HSV-tk gene expression being regulated by TAR-element was evaluated, and activity of the cancer-specific promoters in the Tat-TAR-system was compared. Co-transfection of the cells with the both constructions led to the tat protein synthesis and its affect the HIV-1 TAR-element. An expression level of HSV-tk gene ensured by the both promoters in the binary system was close to that for strong non-specific cytomegalovirus (CMV) promoter. Enzymatic activity of HSV-tk protein in cells having both elements of Tat-TAR-system was two orders of magnitude higher than that in the cells transfected with HSV-tk gene under control of the cancer-specific promoter. Notably, the effect was independent of p53-status of transfected cells: HSV-tk expression level was almost the same in p53(+) and p53(-) cells. The obtained results show that system may be used for therapy of different cancer types both p53-defective and p53-positive ones inhibiting cancer-specific promoters activity.

Experimental data indicating the polyfunctionality of destabilase, a lysozyme from the salivary gland secretion of the medicinal leech, a unique representative of invertebrate lysozymes, were analyzed. The destabilase combines the properties of endo-s-lysyl-y-glutamyl isopeptidase (D-dimer monomerase), lysozyme, and chitinase and simultaneously is a nonenzymatic antimicrobial agent. The polypeptide sequence of lysozyme destabilase is encoded by a family of three genes (Ds1, Ds2, and Ds3). The ability of the enzyme to hydrolyze endoisopeptide bonds formed by transglutaminases, which are detected in many pathological conditions, including thrombosis, is considered from the viewpoint of its further application in practice.

Lipids represent 20% of the total weight of dried pool of medicinal leech salivary gland secretion (SGS) received from about 50 individual animals. There were detected steroids, but not pospholipids in SGS lipid fraction. Immunochemiluminescent analysis identified free steroid hormones in SGS: cortisol, progesterone, testosterone, estradiol and dehydroepiandrosterone. Microchromatografic-mass-spectrometric analysis of SGS and of low-molecular weight fraction (LMW) (molecular masses ranged from 220 to 850 Da) has shown the multicomponent nature of the LMW fraction. Using standart preparations as the reference steroid hormones (cortisol, dehydroepiandrosterone, androstenedione and testosterone) and histamine and serotonine have been identified in SGS.

The protein and peptide composition of medicinal leech salivary gland secretion (SGS) was analyzed in preparations obtained in July from three species--Hirudo verbana, H. medicinalis, and H. orientalis. Two-dimensional electrophoresis (molecular mass 10-150 kD and pI 3-10) revealed no distinctions in the distribution of over 100 silver-stained proteins. Differences were noted only in intensity of 10 protein spots at 30-90 kD and pI 4.7-7.5. Mass spectrometric profiling of SGS of the three leech species using the Zip-Tip/golden chip scheme and cation-exchanging chips CM-10 revealed over 50 components in SGS of each of the three leech species. It was noted that 30-40% of the individual masses of the SGS of each leech species fall within the masses present in SGS of at least one of the two other species. This rather small part of the total mass may be indicative of a high polymorphism of amino acid sequences or a high frequency of posttranslational modifications of the SGS proteins and peptides. Calculation of Jacquard's coefficient showed that H. medicinalis and H. orientalis are closest to each other in SGS composition, which is consistent with data in the literature on the phylogenetic relationship between these two species of medicinal leech. Comparison of detected molecular masses with those of six known biologically active compounds produced by medicinal leeches revealed their uneven distribution in SGS of each of the three medicinal leech species. This opens prospects for using certain species of medicinal leech for targeted therapy of various pathologies.

Secretory polymeric immunoglobulins (IgA dimers and IgM pentamers) are unique in that, apart from L- and H-chains, they contain J-chains responsible for their oligomerization. These antibodies are part of the local adaptive immune system acting on mucosa membranes of the respiratory and digestive systems as the first protection barrier to potential infectious agents. Secretory polymeric immunoglobulins are produced by highly specific B-cells and actively transported to the surface of mucosa membrane through epithelium cells. Therefore, their synthesis and J-chain content are dependent upon epithelium translocation function and condition that are markedly affected by tumorous transformation. Here, we used RT-PCR and immunoblotting to study of the J-chain content and its mRNA expression level in normal and tumorous tissues in lung squamous cell cancer and adenocarcinoma at various stages of disease progression.

Salivary gland secretion (SGS) of the medicinal leech Hirudo medicinalis in summer and winter was studied by proteomic analysis methods, and season-associated difference was found in the distribution of fractionated proteins with the same pattern of their positions. Differences were detected for proteins with molecular weights from 15 to 250 kD fractionated by two-dimensional SDS-PAGE and for 2-10- and 10-60-kD proteins analyzed by SELDI-MS. Thirty-two and 20 proteins were detected by MALDI-TOF-MS in the high-molecular-weight fraction of the summer and winter SGS, respectively, isolated from the corresponding two-dimensional electrophoregrams, and this was less than 20% of the total SGS protein. The N-terminal amino acid sequences were determined for 12 proteins. The peptide maps and N-terminal amino acid sequences of the proteins studied were identified, and no known proteins were revealed. These findings suggest a high content of newly revealed proteins in SGS of medicinal leech, and this correlates with multiple positive clinical effects of hirudotherapy realized through SGS, but the mechanisms of these effects remain unclear.

Background: Since bactericidal properties of some lysozymes are independent of their glycosidase activity, we have investigated this phenomenon for destabilase-lysozyme (DL) from medicinal leech (Hirudo medicinalis)., Methods: Glycosidase activity was determined on Micrococcus luteus, non-enzymatic antibacterial activity of heat-treated DL and of synthetic peptides alpha1, alpha2 and alpha3 (fragments of its primary structure) on M. luteus, Escherichia coli, Bacillus brevis and Streptomyces chrysomallus., Results: Glycosidase activity disappeared after the heating of native DL at 100 degrees C for 40 min. Antibacterial activity of heat-treated DL for M. luteus MDMSU128 and MDMSU140 expressed as minimal inhibitory concentration was 9.8.10(-8) and 12.10(-8) M, respectively, and to E. coli MDMSU52 11.10(-8) M. Antibacterial activity of synthetic peptide alpha1 for M. luteus MDMSU128 and for E. coli MDMSU52 was 8.3.10(-5) and 4.9.10(-5) M, respectively., Conclusion: DL is the first invertebrate lysozyme with combined enzymatic and non-enzymatic antibacterial action.

It is firstly showed that the medicinal leech salivary gland secretion (SGS) as a polycomponent system of proteins and low-molecular weight substances, activates rat subcutaneous mast cells in vitro prompting a decrease in the heparin saturation index and increasing some characteristic mast cells morphometric parameters. The same mast cell changes were detected by analysis of some specimens of subcutaneous cellular tissue in the point of skin injured by the leech bite. It is shown that these changes are saved during 3 days. The mechanical injury of rat skin does not effect the mast cells activation. Activation of mast cells by SGS is extended to the distant subcutaneous mast cells. It is expressed in sharp decreasing of heparin saturation index although not statistically positive. The secondary leeching on these distant points provokes reduction of mast cells activation and some decrease of post-leeching blood heparin content: 0.154 +/- 0.03 units/ml (n = 10) as compared with post-leeching blood heparin contents analysed from the wound after the primary leeching (0.160 +/- 0.03 units/ml, n = 10). Proceeding from these findings, participation of heparin secreted from activated mast cells in the support of post-leeching bleeding is suggested, the phenomenon which provides unloading of capillary pool by application of medicinal leeches for treatment many diseases.

Transient expression of a luciferase reporter gene was used to evaluate tissue-specific promoter and enhancer activities of a solitary extraviral long terminal repeat (LTR) of the human endogenous retrovirus K (HERV-K) in several human and CHO cell lines. The promoter activity of the LTR varied from virtually not detectable (GS and Jurkat cells) to as high as that of the SV40 early promoter (Tera-1 human testicular embryonal carcinoma cells). The negative regulatory element (NRE) of the LTR retained its activity in all cell lines where the LTR could act as a promoter, and was also capable of binding host cell nuclear proteins. The enhancer activity of the LTR towards the SV40 early promoter was detected only in Tera-1 cells and was not observed in a closely related human testicular embryonal carcinoma cell line of different origin, NT2/D1. A comparison of proteins bound to central part of the LTR in nuclear extracts from Tera-1 and NT2/D1 by electrophoretic mobility shift assay revealed striking differences that could be determined by different LTR enhancer activities in these cells. Tissue specificity of the SV40 early promoter activity was also revealed.

Destabilase-lysozyme (DL) from salivary gland secretion of the medicinal leech (Hirudo medicinalis) is as a member of the invertebrate lysozyme family, which sharply differs from other lysozyme families. In this study, DL lysozyme function was confirmed during expression of a gene encoding DL in Escherichia coli. Several constructs of the expression vectors pKK OmpA and pET-3A with or without bacterial, leech, or yeast signal peptides (SP) were used. The use of a construct without signal peptide genes resulted in normal growth of the transformed cells. Transformation of E. coli cells with the constructs containing SP was accompanied by the disruption of the forming cells. The use of the expression vector pET-32 LTC-System for production of DL as a fusion protein with thioredoxin also resulted in normal cell growth. However, specific activity of DL isolated from such cells was significantly lower than that of enzyme purified from extracts of Spodoptera frugiperda cells, which were infected with the baculovirus vector carrying DL cDNA. It is shown that the action mechanism of invertebrate lysozyme does not differ from that of other families: recombinant DL from S. frugiperda extracts catalyzed cleavage of synthetic substrate, hexamer of N-acetylglucosamine, to di- and tetramers, which is typical for enzymatic function of other lysozyme families.

Protein diversity of the high molecular weight fraction (molecular mass > 500 daltons) of salivary grand secretion of the medicinal leech Hirudo medicinalis has been demonstrated using methods of proteomic analysis. One-dimensional (1D) electrophoresis revealed the presence of more than 60 bands corresponding to molecular masses ranging from 11 to 483 kD. 2D-electrophoresis revealed more than 100 specific protein spots differing in molecular masses and pI values. SELDI-mass spectrometry analysis using the ProteinChip. System based on chromatography surfaces of strong anion or weak cation exchanger detected 45 individual compounds of molecular masses ranged from 1.964 to 66.5 kD. Comparison of SELDI-MS data with protein databases revealed eight known proteins from the medicinal leech. Other masses detected by proteomic analytical methods may be related to both modifications of known proteins and unknown biologically active components of leech saliva secretion.

Earlier, three genes Ds1, Ds2, and Ds3 encoding corresponding destabilase-lysozyme isoforms were identified. However only one form of the enzyme encoded by Ds3 gene coincided with the protein CNBr fragments [Mol. Gen. Genet. 253 (1996) 20]. In this work we found by ESI-TOF mass spectrometry that the enzyme preparation consists of at least three forms with molecular masses of 12677.6, 12839.7, and 12938.2Da, each of which contains seven disulfide bridges. Only one mass (12839.7Da) fits to the calculated mass for the protein encoded by Ds3 gene. Further analysis of the CNBr fragments of the enzyme showed the heterogeneity of large 5.5 kDa peptide at positions 64 (threonine or arginine) and 67 (histidine or arginine) in the wild-type amino acid sequence. One CNBr peptide, with Arg and His at positions 64 and 67, respectively, correlates in the molecular mass with the protein encoded by Ds3. In addition, we have found a new acid form of destabilase-lysozyme, P-Ac, which differs from all known destabilase-lysozyme structures by its N-terminal amino acid sequence.

The effects of components of the salivary gland secretion (proteases and protease inhibitors) of the medicinal leech (Hirudo medicinalis) on the growth of neurites of sensory neurons from chick embryos (10-11 days old) were studied in organotypic cultures. Destabilase and high-molecular-weight bdellin B, (0.01, 0.02, 0.05, and 0.1 ng/ml), bdellastasin (0.02 and 0.05 ng/ml), and eglin C (0.1 ng/ml) had neurite-stimulating effects on day 3 of cultivation of spinal ganglia. Identification of the neurite-stimulating activity of these components of medicinal leech salivary gland secretions creates the basis for creating new therapeutic agents for the treatment of neurodegenerative diseases.

The effects of components from medicinal leech (Hirudo medicinalis) salivary gland secretions and the therapeutic agent Piyavit on the growth of chick embryo neurites in organotypic culture were studied. Native destabilase and bdellin A at concentrations of 0.01, 0.02, 0.05, and 0.1 ng/ml, bdellin B at a concentration of 0.05 ng/ml, and eglin at a concentration of 0.1 ng/ml had neurite-stimulating activity, evident on the third day of organotypic culture of spinal ganglia. The stimulatory activity of destabilase was lost after revere-phase chromatography. The neurite-stimulating activity of the extract of the therapeutic agent Piyavit (200 ng/ml) in organotypic ganglion culture appeared to result from the neurite-stimulating salivary gland components within this agent, suggesting that Piyavit could be used for the treatment of neurodegenerative disorders.

A novel affinity elution procedure for purification of DNA-binding proteins was developed and employed to purify to near homogeneity the proteins recognizing a 21 base pair sequence within the long terminal repeat of human endogenous retroviruses K. The approach involves loading the initial protein mixture on a heparin-agarose column and elution of protein(s) of interest with a solution of double-stranded oligonucleotide containing binding sites of the protein(s). The affinity elution has several advantages over conventional DNA-affinity chromatography: (i) it is easier and faster, permitting to isolate proteins in a 1 day-one stage procedure; (ii) yield of a target protein is severalfold higher than that in DNA-affinity chromatography; (iii) it is not necessary to prepare a special affinity support for each factor to be isolated. Theaffinity elution could be a useful alternative to conventional DNA-affinity chromatography.

Thrombin inhibitors from bloodsucking leeches and insects that block conversion of fibrinogen to fibrin are considered. Regulatory mechanisms influencing the fibrinogen-fibrin system in leeches include fibrinogen degradation, inhibition of factor XIIIa, and lysis of fibrin clots. The review also summarizes recent data on plasminogen activator from the vampire bat Desmodus rotundus and a role of fibrin as its cofactor.

Saliva of bloodsuckers (leeches, insects, ticks, vampire bats) contains various regulators of some hemostatic stages. This review summarizes information on their structural characteristics and mechanisms of action. Most bloodsuckers are shown to inhibit vascular-platelet hemostasis by blocking collagen-induced platelet adhesion/aggregation. Plasma hemostasis is inhibited by blocking activation of factor X or factor Xa directly.

Destabilase, endo-epsilon-(gamma-Glu)-Lys-isopeptidase, was prepared from the salivary gland secretion of the medicinal leech (Hirudo medicinalis). The secretion prepared by the known method of Rigbi et al. (1987) (secretion-K) lacks the destabilase-characteristic highly specific isopeptidase activity (the D-dimer-monomerizing activity) because of its degradation by proteolytic activity (the substrate of Glp-Ala-Ala-Leu-pNA) due to contamination with leech intestinal channel contents. Therefore, we have elaborated a new technique for preparation of a true leech secretion (secretion-I). This secretion is characterized by the complete absence of the leech intestinal channel contents and has no proteolytic activity. For the first time the destabilase-specific D-dimer-monomerizing and lysozyme activities were separated by fractionation of secretion-I by HPLC gel filtration through Superose S-12. For the purified destabilase preparation, these activities were separated by reversed-phase chromatography in an acetonitrile gradient (0-60%) in the presence of 0.1% trifluoroacetic acid. The monomerizing activity of destabilase is responsible for the ability of secretion-I to dissolve stabilized fibrin via isopeptidolysis of alpha-alpha and gamma-gamma fibrin chains bound by epsilon-(gamma-Glu)-Lys-isopeptide bonds.

Components of leech (Hirudo medicinalis) salivary gland secretion (high molecular mass bdelin B, bdellastasin, eglin C, destabilize) were used to investigate their neurotrophic effect on organotypic culture of dorsal root ganglia of the 10-11-day old chicken embryos. Destabilize and high molecular mass bdelin B in concentrations of 0.01, 0.02, 0.05 and 0.1 ng/ml, bdellastasin in concentrations of 0.02 and 0.05 ng/ml, eglin C in concentrations of 0.1 ng/ml exhibit neurite-stimulating effect as compared to the control.

The medicinal leech Hirudo medicinalis produces various types of proteinase inhibitors: bdellins (inhibitors of trypsin, plasmin, and acrosin), hirustasin (inhibitor of tissue kallikrein, trypsin, alpha-chymotrypsin, and granulocyte cathepsin G), tryptase inhibitor, eglins (inhibitors of alpha-chymotrypsin, subtilisin, and chymasin and the granulocyte proteinases elastase and cathepsin G), inhibitor of factor Xa, hirudin (thrombin inhibitor), inhibitor of carboxypeptidase, and inhibitor of complement component C1s. This review summarizes data on their primary and tertiary structures, action mechanisms, and biological activities.

Effects of destabilise, bdellin, bdellin A, eglin were investigated in organotypic tissue culture of dorsal root ganglia (DRG) of 10-11-day old chick embryos. Native destabilase and bdellin A, bdellin B and eglin are more active inducing a more intensive neurite growth in DRG as compared with the control. A neurite-stimulating effect of the drug "pyjavit" seems to be associated with destabilase, bdellins and eglin neurite-stimulating activity.

In the paper are presented the results of the study of Pyavit, a complex leech preparation from, whose effects on the memory may be dependent on DNA methylation (one of the mechanisms of gene expression regulation). A positive effect of Pyavit on formation and retention of passive avoidance conditioned reaction was demonstrated in rat experiments.

Intrinsic lysozyme-like activity was demonstrated for destabilase from the medicinal leech supported by (1) high specific lysozyme activity of the highly purified destabilase, (2) specific inhibition of the lysozyme-like activity by anti-destabilase antibodies, and (3) appreciable lysozyme-like activity in insect cells infected with recombinant baculoviruses carrying cDNAs encoding different isoforms of destabilase. Several isoforms of destabilase constitute a protein family at least two members of which are characterized by lysozyme activity. The corresponding gene family implies an ancient evolutionary history of the genes although the function(s) of various lysozymes in the leech remains unclear. Differences in primary structures of the destabilase family members and members of known lysozyme families allow one to assign the former to a new family of lysozymes. New proteins homologous to destabilase were recently described for Caenorhabditis elegans and bivalve mollusks suggesting that the new lysozyme family can be widely distributed among invertebrates. It remains to be investigated whether the two enzymatic activities (isopeptidase and lysozyme-like) are attributes of one and the same protein.

A study was made of the dependence of the hydrolysis rate of isopeptide bonds in the D-dimer, the product of limited proteolysis of the stabilized fibrin by plasmin, on the high-molecular substrate concentration catalyzed by destabilase from the medicinal leech. The formation of the reaction product was electrophoretically measured by the increase in the quantity of D-monomer. This dependence was shown to be nonlinear.

The effect of destabilase, a component of Hirudo medicinalis salivary gland secret, was investigated in organotypic tissue culture of dorsal root ganglia (DRG) of 10-11-day old chick embryos. Native destabilase in concentrations 0.01 and 0.05 ng/ml was active, inducing a more intensive neurite growth in DRG that in the control. The stabilizing activity of destabilase was lost following reverse-phase chromatography. Neurite-stimulating effects of the drug "pyjavit" is due presumably to neurite-stimulating activity of destabilase.

The protective antithrombotic effects of piyavit, a novel pharmacological preparation from the medicinal leech, its water extract concentrated and diluted 1600-fold, the piyavit fraction containing leech prostanoids and highly purified destabilase, epsilon-(gamma-Glu)-Lys-isopeptidase isolated from the medicinal leech (Hirudo medicinalis), have been studied. All the preparations under study inhibited clot formation in rat mesanterium arterioles stimulated by laser energy, contained leech prostanoids immunoenzymatically identified as 6-keto-PgF1 alpha and inhibited platelet aggregation induced by ADP. The putative mechanism of their antithrombotic effect is discussed.

Piyavit, the novel pharmacological preparation from the medicinal leech (Hirudo medicinalis) contained the leech saliva, produces the potent arterial antithrombotic effect examined on experimental of Laser Induced Thrombus formation. Administrated orally into rats or injected subcutaneously as water extract, non-diluted or in 1600 times diluted, it inhibits statistically significant comparing with control platelet thrombus stimulated by laser beams. Its components, prostanoid fraction and purified enzyme destabilase, endo-epsilon-(gamma-Glu)-Lys-isopeptidase, also inhibit thrombus formation in statistically different manner, comparing with control. All the tested preparations inhibit platelet aggregation induced by ADP. The dependence of arterial antithrombotic effect on the leech prostanoid is discussed.

The highly specific enzyme--destabilase [endo-epsilon-(gamma-glutamyl)- lysyl-isopeptidase]--similar to the true protease, acquires after incubation with sodium dodecyl sulfate a pronounced proteolytic activity towards certain proteins. At the same time, the amidolytic activity of the enzyme (substrate of p-nitroanilide gamma-glutamic acid) disappears, while its ability to catalyse the monomerization of the D-D dimer, a fragment of stabilized fibrin, is preserved. After SDS removal by gel filtration, the enzyme loses its nonspecific proteolytic activity, while its specific activity is fully reconstituted. Other detergents produce a more weak influence upon the enzyme. The putative mechanism providing the increase in the enzyme specificity in the presence of sodium dodecyl sulfate is discussed.

Catalytic activity of destabilase has been studied in hydrolyses of synthetic and natural substrates. High specificity of the enzyme is shown in hydrolysis of endo-epsilon-(gamma-Glu)Lys isopeptide bonds.

The salivary gland secretion of the medicinal leech catalyzes the conversion into monomeric form of the fragment D dimer, the product of limited proteolysis of stabilized fibrin. Analysis of N-terminal sequences revealed identical fragments for the D-dimer gamma-gamma-chains and the D-monomer gamma-chains formed via this reaction and established the presence of only one N-terminal amino acid (Ser). These results provide evidence for the preservation of integrity of the polypeptide chains during monomerization of the D-dimer.

The molecular mass of destabilase isolated from the medicinae leech Hirudo medicinalis was found to be equal to 12.3 kDa. A kinetic analysis of the sole presently known synthetic substrate, L-gamma-Glu-pNA, showed that the enzyme is relatively stable to heating (5 min, 70 degrees C); the pH optimum lies at 7.0-8.5. The enzyme has a specific activity of 0.15 x 10(-9) mol.s-1.mg-1; Km = 2.2 x 10(-4) M, kcat is 3.53 x 10(-3) s-1 (pH 8.0, 37 degrees C).

New analogues of cAMP, namely 2'-phosphate of cAMP and its esters, were obtained by direct phosphorylation of N6-benzoyladenosine 3',5'-cyclic phosphate with 2-cyanoethyl phosphate in the presence of DCC followed by the removal of protecting groups.