Thrombolytic agents of the preparation from the medicinal leeches

The thrombolytic effect of an extract from medicinal leeches orally administered to rats was shown. This effect depends on the number of administrations and on the concentration of extract. It is due to the leech prostaglandins and enzyme destabilase. After the extraction of prostaglandins by ethylacetate the thrombolytic effect diminished by 45%. It is supposed that the leech-prostaglandins, like prostacyclin and its stable analogous, induce the release of tissue plasminogen activator from vessel walls. The thrombolytic effect of destabilase was demonstrated in experiments in vitro. We observed the phenomenon of increasing of glutaminase activity of citrate blood and the appearance of amidolytic and destabilase activity.     

Isolation and complete amino-acid sequence of the small polypeptide from light-harvesting pigment-protein complex I (B870) of Rhodopseudomonas capsulata

The small bacteriochlorophyll-binding polypeptide of the light-harvesting complex B870 was extracted from the intracytoplasmic membrane of the strain A1a+ of Rhodopseudomonas capsulata with chloroform/methanol/ammonium acetate and separated by chromatography on Sephadex LH60 using the same solvent. The polypeptide obtained from the peak fraction III was found to be homogeneous and identical with the small polypeptide isolated from the B870 complex as shown by dodecyl sulfate/polyacrylamide gel electrophoresis, amino acid composition and N-terminal sequence. The complete amino acid sequence is given. The relative molecular mass based on the amino acid sequence is 5341. The polarity of amino acids is 35.42%. The C-terminal part of the peptide chain from residue 29 to 48 is hydrophobic and includes one His residue.     

Recombinant Destabilase from Hirudo medicinalis Is Able to Dissolve Human Blood Clots In Vitro

Leeches are amazing animals that can be classified as conditionally poisonous animals since the salivary cocktail they produce is injected directly into the victim, and its components have strictly defined biological purposes, such as preventing blood clot formation. Thrombolytic drugs are mainly aimed at treating newly formed blood clots. Aged clots are stabilized by a large number of isopeptide bonds that prevent the action of thrombolytics. These bonds are destroyed by destabilase, an enzyme of the leech’s salivary glands. Here, we conducted a pilot study to evaluate the feasibility and effectiveness of the use of destabilase in relation to blood clots formed during real pathological processes. We evaluated the isopeptidase activity of destabilase during the formation of a stabilized fibrin clot. We showed that destabilase does not affect the internal and external coagulation cascades. We calculated the dose–response curve and tested the ability of destabilase to destroy isopeptide bonds in natural blood clots. The effect of aged and fresh clots dissolving ability after treatment with destabilase coincided with the morphological characteristics of clots during surgery. Thus, recombinant destabilase can be considered as a potential drug for the treatment of aged clots, which are difficult to treat with known thrombolytics.     

Polyfunctionality of destabilase, a lysozyme from a medicinal leech

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.     

The mushroom Marasmius oreades lectin is a blood group type B agglutinin that recognizes the Galalpha 1,3Gal and Galalpha 1,3Galbeta 1,4GlcNAc porcine xenotransplantation epitopes with high affinity

A blood group B-specific lectin from the mushroom Marasmius oreades (MOA) was investigated with respect to its molecular structure and carbohydrate binding properties. SDS-PAGE mass spectrometric analysis showed it to consist of an intact (H; 33 kDa) and truncated (L; 23 kDa) subunit in addition to a small polypeptide (P; 10 kDa). Isolation in the presence of EDTA produced only the H subunits, indicating that the latter two are formed by metalloprotease cleavage of the intact H subunit. Tryptic digestion of the H, L, and P polypeptide chains followed by mass spectral analysis supports this view. The lectin strongly precipitated blood group type B substance, was nonreactive with type A substance, and reacted weakly with type H substance. Carbohydrate binding studies reveal a high affinity for Galalpha1,3Gal (but not for the isomeric alpha1,2-, alpha1,4-, and alpha1,6-disaccharides); Galalpha1,3Galbeta1,4GlcNAc; and the type B branched trisaccharide. MOA also reacts strongly with murine laminin from the Engelbreth-Holm-Swarm sarcoma and bovine thyroglobulin, both of which contain multiple Galalpha1,3Galbeta1,4GlcNAc end groups. This linear B trisaccharide is a component of porcine tissues and organs, preventing their transplantation into humans. MOA also shares carbohydrate recognition of this trisaccharide with toxin A elaborated by Clostridium difficile.     

A molecular dynamics study of the stoichiometric complex formed by poly (alpha, L-glutamate) and octyltrimethylammonium ions in chloroform solution.

We present a molecular dynamics simulation at 300 K in explicit solvent environment of chloroform of the stoichiometric complex formed by poly(alpha,L-glutamate) and octyltrimethylammonium ions. We observed that the alpha-helix conformation of the polypeptide chain remains stable during a 2-ns run. The surfactant ions predominantly adopted an extended conformation that is stabilized by favorable interactions with the organic solvent. Analysis of the organization of the surfactant with respect to the polypeptide chain indicated that each octyltrimethylammonium cation was preferentially bound to more than one carboxylate group. It was found that the most populated arrangement was that with the surfactant cations interacting with two carboxylate groups simultaneously.     

Polyfunctionality of lysozyme destabilase from the medicinal leech

Experimental data indicating the polyfunctionality of lysozyme destabilase from the salivary gland secretion of the medicinal leech, a unique representative of invertebrate lysozymes, were analyzed. The destabilase combines the properties of endo-?-lysyl-γ-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 under many pathological conditions, including thrombosis, is considered from the viewpoint of its further application in practice.     

Molecular dynamics simulations of a winter flounder “antifreeze” polypeptide in aqueous solution

A winter flounder antifreeze polypeptide (HPLC-6) has been studied in vacuo and in aqueous solution using molecular dynamics computer simulation techniques. The helical conformation of this polypeptide was found to be stable both in vacuum and in solution. The major stabilizing interactions were found to be the main-chain hydrogen bonds, a salt-bridge interaction, and solute–solvent hydrogen bonds. A significant bending in the middle of the polypeptide chain was observed both in vacuo and in solvent at 300 K. Possible causes of the bending are discussed. From simulations of mutant polypeptide molecules in vacuo, it is concluded that the bend in the native polypeptide was caused by side chain to backbone hydrogen bond competition involving the Thr 24 side chain and facilitated by strains on the helix resulting from the Lys 18-Glu 22 salt bridge. © 1993 John Wiley & Sons, Inc.     

Molecular cloning of subunits of complex I from Neurospora crassa. Primary structure and in vitro expression of a 22-kDa polypeptide

A lambda gt11 cDNA expression library was screened with antibodies directed against individual subunits of complex I from Neurospora crassa. Clones encoding cytoplasmically synthesized polypeptides with apparent molecular masses of 22, 29, 31, and 33 kDa were isolated. Northern blot analysis revealed that the corresponding genes are transcribed into mRNA species of about 0.85, 0.95, 1.3, and 1.4 kilobases, respectively. Further characterization of clones encoding the 22-kDa subunit was performed. A cDNA insert of 755 base pairs containing the complete coding sequence was used to express the polypeptide in vitro. A precursor of the protein is synthesized on cytoplasmic ribosomes without a cleavable signal sequence. Our data indicate that after import into the organelle and before assembly into complex I, the 22-kDa polypeptide forms intramolecular disulfide bridge(s). Nucleotide sequencing revealed an open reading frame coding for a protein of 183 amino acids. A molecular mass of 20,828 daltons was calculated. The polypeptide is hydrophilic and contains no obvious membrane-spanning domains. Eight cysteine residues arranged in a regular pattern are found in the primary structure of the protein. Therefore, this subunit is a good candidate to bind at least one of the iron-sulfur centers present in complex I of the respiratory chain.     

Studies on cytochrome c oxidase, IX. The primary structure of polypeptide VIa

The complete amino acid sequence of the cytoplasmic polypeptide VIa of cytochrome c oxidase from beef heart is described. The primary structure of this component of complex IV of the respiratory chain is elucidated by isolation and sequencing of overlapping glutamic acid, arginine, tryptophan and methionine fragments obtained by cleavage with Staphylococcus aureus protease, protease from submaxillaris glands of mice, 2-iodosylbenzoic acid and cyanogen bromide. The chain length of polypeptide VIa is 98 amino acids, the resulting molecular mass of 10670 Da. The hydrophilic protein does not contain a hydrophobic membrane penetrating sequence domain. Its function in the respiratory complex IV is unknown.     

Evidence for a single-chain structure of native human ceruloplasmin using sodium dodecyl sulfate-polyacrylamide-crossed immunoelectrophoresis

A simple method is described for the analysis of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with crossed immunoelectrophoresis. The technique allows the identification of specific proteins and estimation of their molecular weights after the electrophoresis of complex mixtures such as blood plasma. Human ceruloplasmin was estimated to have a molecular weight of 128,000 using this method. Reduction of plasma with 2-mercaptoethanol in the presence of protease inhibitors did not result in dissociation of ceruloplasmin into detectable smaller subunits, suggesting that native ceruloplasmin is composed of a single polypeptide chain.     

The effects of alcohols on the structure of human glycophorin

The effects of alcohols on human glycophorin were monitored by circular dichroism, solvent perturbation of absorption spectra, fluorescence of 8-anilino-1-naphthalene sulfonate, and sedimentation equilibrium in the ultracentrifuge. Both ethanol and 2-chloroethanol gradually increase the alpha helix in glycophorin and its sialic acid free counterpart. The same alcohols do not cause a cooperative transition in the structure of the polypeptide chain of glycophorin. Other alcohols also increase the alpha-helix content of glycophorin. Binding of ANS to glycophorin is abolished at relatively low alcohol concentrations. Ethanol at 60% (v/v) reduces the molecular weight ratio of glycophorin and at the same time increases the exposure of tyrosine residues to solvent. These observations indicate a complex mechanism of interaction of weakly protic solvents with this stable membrane protein.     

Site-directed mutagenesis and 1H NMR spectroscopy of an interdomain segment in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

Deletion of two of the three homologous lipoyl domains that form the N-terminal half of each dihydrolipoamide acetyltransferase (E2p) polypeptide chain of the Escherichia coli pyruvate dehydrogenase complex can be achieved by in vitro deletion in the structural gene aceF. A site-directed mutagenesis of this shortened aceF gene was carried out to replace the glutamine residue at position 291 (wild-type numbering) with a histidine residue. Residue 291 is near the middle of a long segment (about 30 amino acid residues) of polypeptide chain, rich in alanine, proline, and charged amino acids, that links the remaining lipoyl domain to the dihydrolipoamide dehydrogenase (E3) binding domain in the E2p chain. A fully active enzyme complex was still assembled, and despite the enormous size of the particle (Mr approximately 4 x 10(6)), sharp resonances attributable to the single new histidine residue per E2p chain could be detected in the 400-MHz 1H NMR spectrum of the complex. The sharpness of these resonances, their chemical shifts (7.94 and 7.05 ppm), and the apparent pKa (6.4) of the side chain were all consistent with this histidine residue being exposed to solvent in a conformationally flexible region of the E2p polypeptide chain. These experiments provide direct proof for the conformational flexibility of this region of polypeptide chain, which is thought to play an important part in the movement of the lipoyl domain required for active site coupling in the enzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant destabilase from the medicinal leech: Preparation and properties

The procedure for obtaining an active recombinant destabilase from the medicinal leech in Escherichia coli cells was developed. The plasmids encoding an analogue of native destabilase, as well as the protein forms carrying polyhistidine sequence at the Cand/or N-terminus of the polypeptide were obtained during the work. The producing strains of different forms of the protein were constructed, the cultivation process was optimized. The conditions of renaturation of destabilase recombinant forms by dialysis and using chromatographic absorbent were selected. The muramidase activity towards cell walls of Micrococcus lysodeikticus bacferia and lytic activity towards E. coli were investigated. The dependence of pH and ionic strength of the solution on the activities was determined. The total antibacterial activity of destabilase towards E. coli was shown.     

Molecular basis for the effect of urea and guanidinium chloride on the dynamics of unfolded polypeptide chains

Chemical denaturants are frequently used to unfold proteins and to characterize mechanisms and transition states of protein folding reactions. The molecular basis of the effect of urea and guanidinium chloride (GdmCl) on polypeptide chains is still not well understood. Models for denaturant--protein interaction include both direct binding and indirect changes in solvent properties. Here we report studies on the effect of urea and GdmCl on the rate constants (k(c)) of end-to-end diffusion in unstructured poly(glycine-serine) chains of different length. Urea and GdmCl both lead to a linear decrease of lnk(c) with denaturant concentration, as observed for the rate constants for protein folding. This suggests that the effect of denaturants on chain dynamics significantly contributes to the denaturant-dependence of folding rate constants for small proteins. We show that this linear dependency is the result of two additive non-linear effects, namely increased solvent viscosity and denaturant binding. The contribution from denaturant binding can be quantitatively described by Schellman's weak binding model with binding constants (K) of 0.62(+/-0.01)M(-1) for GdmCl and 0.26(+/-0.01)M(-1) for urea. In our model peptides the number of binding sites and the effect of a bound denaturant molecule on chain dynamics is identical for urea and GdmCl. The results further identify the polypeptide backbone as the major denaturant binding site and give an upper limit of a few nanoseconds for residence times of denaturant molecules on the polypeptide chain.     

A chlorophyll-protein complex lacking in photosystem I mutants of Chlamydomonas reinhardtii

Sodium dodecyl sulfate gel electrophoresis of unheated, detergent-solubilized thylakoid membranes of Chlamydomonas reinhardtii gives two chlorophyll-protein complexes. Chlorophyll-protein complex I (CP I) is the blue-green in color and can be dissociated by heat into "free" chlorophyll and a constituent polypeptide (polypeptide 2; mol wt 66,000). Similar experiments with spinach and Chinese cabbage show that the higher plant CP I contains an equivalent polypeptide but of slightly lower molecular weight (64,000). Both polypeptide 2 and its counterpart in spinach are soluble in a 2:1 (vol/vol) mixture of chloroform-methanol. Chemical analysis reveals that C. reinhardtii CP I has a chlorophyll a to b weight ratio of about 5 and that it contains approximately 5% of the total chlorophyll and 8-9% of the total protein of the thylakoid membranes. Thus, it can be calculated that each constituent polypeptide chain is associated with eight to nine chlorophyll molecules. Attempts to measure the molecular weight of CP I by calibrated SDS gels were unsuccessul since the complex migrates anomalously in such gels. Two Mendelian mutants of C. reinhardtii, F1 and F14, which lack P700 but have normal photosystem I activity, do not contain CP I or the 66,000-dalton polypeptide in their thylakoid membranes. Our results suggest that CP I is essential for photosystem I reaction center activity and that P700 may be associated with the 66,000-dalton polypeptide.     

X-ray small-angle studies of the pyruvate dehydrogenase core complex from Escherichia coli K-12

The pyruvate dehydrogenase core complex from E. coli K-12, defined as the multienzyme complex which can be obtained with a unique polypeptide chain composition, has been investigated in solution with the X-ray small-angle technique. The molecular mass of the core complex of 3.78·10⁶ daltons verifies the ratio of polypeptide chains of 161616 of the three enzyme components, pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, present in the complex. In connection with the values obtained for the radius of gyration (156.5 å), volume (1.07·10⁷ å³) and amount of solvent associated with the complex (1.03 g/g) a loose packing of subunits in the complex has to be assumed. The maximum diameter of the core complex of 433 å, as determined from the correlation function, corroborates the large extension of the complex. The comparison of experimental and theoretical scattering curves reveals a relatively isometric overall shape of the core complex.Enzymes: Pyruvate dehydrogenase complex = pyruvate dehydrogenase (EC 1.2.4.1) plus dihydrolipoamide transacetylase (EC 2.3.1.12) plus dihydrolipoamide dehydrogenase (EC 1.6.4.3).     

CONTROL OF GRANULOCYTE PRODUCTION: SEPARATION AND CHEMICAL IDENTIFICATION OF A SPECIFIC INHIBITOR (CHALONE)

Abstract. It has previously been shown by others that blood serum contains inhibitors of blood cell production acting on the proliferation of granulocy tic and erythrocytic precursor cells in the bone marrow. It is now shown that the active extract from calf blood serum can be further subfractionated into six different components, all of them exhibiting inhibitory effects on the proliferation of rat bone marrow cells in vitro. Ascitic fluid from rats treated intraperitoneally with polyvinylpyrrolidone contains inhibitors which apparently are the same as those found in calf serum.
It was further possible to demonstrate that only one of these inhibitors is contained in mature granulocytes where it is actively synthesized from amino acids and subsequently released into the surrounding medium. By chromatography on Sephadex G-25 of this conditioned medium the inhibiting substance could be obtained in relatively pure form being contaminated only by low amounts of two ninyhdrin-positive substances. the experiments allow the granulocytic inhibitor to be identified as a polypeptide with a molecular weight below 5000. the results suggest that this substance is the granulocytic chalone.     

Modeling the structure of agitoxin in complex with the Shaker K+ channel: a computational approach based on experimental distance restraints extracted from thermodynamic mutant cycles

Computational methods are used to determine the three-dimensional structure of the Agitoxin (AgTx2)-Shaker complex. In a first stage, a large number of models of the complex are generated using high temperature molecular dynamics, accounting for side chain flexibility with distance restraints deduced from thermodynamic analysis of double mutant cycles. Four plausible binding mode candidates are found using this procedure. In a second stage, the quality and validity of the resulting complexes is assessed by examining the stability of the binding modes during molecular dynamics simulations with explicit water molecules and by calculating the binding free energies of mutant proteins using a continuum solvent representation and comparing with experimental data. The docking protocol and the continuum solvent model are validated using the Barstar-Barnase and the lysozyme-antibody D1.2 complexes, for which there are high-resolution structures as well as double mutant data. This combination of computational methods permits the identification of two possible structural models of AgTx2 in complex with the Shaker K+ channel, additional structural analysis providing further evidence in favor of a single model. In this final complex, the toxin is bound to the extracellular entrance of the channel along the pore axis via a combination of hydrophobic, hydrogen bonding, and electrostatic interactions. The magnitude of the buried solvent accessible area corresponding to the protein-protein contact is on the order of 1000 A with roughly similar contributions from each of the four subunits. Some side chains of the toxin adopt different conformation than in the experimental solution structure, indicating the importance of an induced-fit upon the formation of the complex. In particular, the side chain of Lys-27, a residue highly conserved among scorpion toxins, points deep into the pore with its positively charge amino group positioned at the outer binding site for K+. Specific site-directed mutagenesis experiments are suggested to verify and confirm the structure of the toxin-channel complex.