Inhibition of the proteolytic activity of thrombin by methyl esters of arginine-containing oligopeptides

The kinetics of thrombin-catalyzed hydrolysis of the esters of arginine-containing di- and tripeptides at pH 8.5 and the inhibition of fibrinogen-thrombin reaction by these compounds are studied. The experimentally obtained values of Km, kcat, I50 and the suggestion about the competitive character of the antithrombin action of the peptides allowed the individual kinetic parameters to be calculated. The data obtained indicate that the efficiency of the thrombin-catalyzed hydrolysis of the peptides or a degree of the retardation of the proteolytic activity of thrombin by the esters under investigation depend on the hydrophobicity of the amino acid residues located at subsides P2 and P3 of the peptides. An increase of the hydrophobicity has induced a decrease in the rate constants k2 and k3 and an increase in the inhibition power of the oligopeptides.     

Kinetics of trypsin-catalyzed hydrolysis of some arginine-containing peptide methyl esters

The kinetics of trypsin-catalyzed hydrolysis (at pH 8.5) of methyl esters of some synthetic dipeptides containing the residues of both arginine and L(D)-p-fluorophenylalanine or L(D)-tyrosine has been studied. The digestion of Tos-(pF)Phe-Arg-OMe was shown as unfollowing the Michaelis-Menten kinetic since in the reaction course the substrate activation is observed and while the reaction product is the enzymatic process inhibitor. In contrast to this, the hydrolysis of other substrates studied, follows the normal Michaelis-Menten kinetic.     

Inhibitory effect of methyl esters of arginine-containing oligopeptides on thrombin and trypsin

Stereoisomeric oligopeptides were studied for their inhibitory effect on the hydrolysis of benzoyl-L-arginine methyl ester catalyzed by thrombin and trypsin, as well as on the thrombin-fibrinogen reaction. Comparison of the peptide structures, their conformational flexibility and inhibitory effects on thrombin and trypsin shows the availability of the essential differences in organization and functioning of the subsites S3, S2 and S'1 of these enzymes. In contrast to trypsin, thrombin is shown to be characterized by more pronounced secondary stereospecificity. This manifests in the more vigorous dropping of the catalytic constants of thrombin-catalyzed esterolysis than those of trypsin-catalyzed hydrolysis of the substrates, containing D-amino acids at the subsite P2. It is revealed that the peptide Tos-D-Val-D-Ala-D-Agr-D-Phe-OCH3 is the most powerful inhibitor among studied compounds. It is noteworthy that its antithrombin effect is almost an order of magnitude higher than its antitrypsin effect.     

Solid-state IR-LD spectroscopic and theoretical analysis of arginine-containing peptides

Structural prediction and IR-characteristic bands assignment of arginine-containing tri- and tetrapeptides glycyl-glycyl-arginine (GGArg) and glycyl-glycyl-arginyl-alanine (GGArgAla) have been carried out, using linear-dichroic infrared (IR-LD) spectroscopy of oriented solid-samples in a nematic liquid crystal suspension. Spectroscopic data have been supported with ab initio analysis (Hartree-Fock level of theory and 6-31++G** basis set). Predicted geometry parameters have been compared with known crystallographic ones of similar peptides, indicating a good correlation.     

Functional arginine-containing amino acid sequences in peptides and proteins

L-Arginine is a source of nitrogen oxide and plays a great role in a number of other biochemical processes. Functions and prospects for practical application of five groups of arginine-containing amino acid sequences and synthetic polyarginine sequences are considered. The physiological characteristics of well-known arginine-containing peptides, such as RGD containing, kyotorphin, and tuftsin, are described in detail.     

Functional arginine-containing amino acid sequences in peptides and proteins

L-arginine is a source of nitrogen oxide and plays a great role in a number of other biochemical processes. Functions and prospects for practical application of five groups of arginine-containing amino acid sequences and synthetic polyarginine sequences are considered. The physiological characteristics of well-known arginine-containing peptides, such as RGD peptides, kyotorphin, and tuftsin, are described in detail. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru     

Synthesis of arginine-containing peptides and their conjugates with protohemin IX and tetraphenylporphyrin

Arg-containing peptides and their conjugates with protohemin IX were synthesized by the solid phase method using Merrifield resin. The conjugates of Arg-containing peptides with tetraphenylporphyrin were obtained by using phosphorus trichloride as an activating agent.     

Correction of experimental metabolic syndrome manifestations in rats by some arginine-containing peptides

Functional characterization of new short glyproline peptides, which are able to provide a regulatory effect on the functional state of the hemostasis system, as well as lipid and carbohydrate metabolism in the body, is an actual task of physiology and medicine. In the present study, we used a model of experimental metabolic syndrome developed in animals due to continuous feeding with high-calorie food. This leads to increased clotting, glucose concentration, low-density lipoprotein cholesterol, triglycerides, and the level of total cholesterol in the blood, which is accompanied by an increase in the body weight of rats. Arginine-containing peptides (Arg-Glu-Arg-Pro-Gly-Pro, Arg-Glu-Arg-Val-Gly-Pro, Arg-Glu-Arg-Gly-Pro) were intranasally administered every 24 h to rats seven times 6 weeks after the development of metabolic syndrome. These peptides provided a unique combined effect on the body, restoring parameters of lipid metabolism, the hemostasis system, and the concentration of blood glucose to normal values. The corrective effect of the studied peptides was detected 20 h after the last administration and was maintained for 168 h even under further feeding of rats with high-calorie diet. The studied glyproline peptides belong to therapeutic normoglycemic and lipid-lowering drugs. They block the accumulation of new fat deposits in the body, and also have anticoagulant and antithrombotic effects in disorders of lipid metabolism. The Arg-Glu-Arg-Pro-Gly-Pro peptide possessed the most pronounced and stable positive effect on the body.     

The C-terminal binding domain of hirullin P18. Antithrombin activity and comparison to hirudin peptides

Hirullin P18 is a 61-amino acid hirudin-related protein having potent antithrombin activity. Similar to hirudin, it contains a highly acidic C-terminus, but has a significantly different sequence from any other known hirudin variant. The present study demonstrates that the C-terminal fragment acetyl-hirullin P18(41-62) [corrected] possesses an antithrombin potency similar to that of acetyl-desulfatohirudin(45-65). Additionally, like the hirudin fragment analog, it inhibits fibrin-clot formation by binding to a non-catalytic site on thrombin. Sequential shortening of the hirullin P18 C-terminal fragment demonstrates the critical nature of Phe51, which corresponds to the important Phe56 residue of hirudin. Although the sequences of hirullin P18(54-61) and hirudin(59-65) have substantial differences, the C-terminal functional domain represented by hirullin P18(50-61) appears to be comparable to hirudin(55-65) in terms of its functional role in antithrombin activity.     

Antimicrobial activity of fatty acid methyl esters of some members of Chenopodiaceae

Fatty acid methyl ester (FAME) extracts of four halophytic plants, viz. Arthrocnemum indicum, Salicornia brachiata, Suaeda maritima and Suaeda monoica belonging to the family Chenopodiaceae, were prepared and their composition was analyzed by GC-MS. The FAME extracts were also screened for antibacterial and antifungal activities. The GC-MS analysis revealed the presence of more saturated fatty acids than unsaturated fatty acids. Among the fatty acids analyzed, the relative percentage of lauric acid was high in S. brachiata (61.85%). The FAME extract of S. brachiata showed the highest antibacterial and antifungal activities among the extracts tested. The other three extracts showed potent antibacterial and moderate anticandidal activities.     

Antithrombin III activity in baboons

The antithrombin III-heparin cofactor activity of 65 baboons and 130 healthy human subjects was measured. The results indicate that antithrombin III-heparin cofactor activity is significantly lower in baboons than in humans. The increased activity of the coagulation system of baboon is coupled with decreased antithrombin III-heparin cofactor activity. In humans each of these changes is associated with increased risk of thrombosis, but the baboon has a very low risk of developing thrombosis. Other factors probably balance the coagulation-coagulation inhibition systems in baboons.     

Towards a structure-function analysis of bovine lactoferricin and related tryptophan- and arginine-containing peptides.

The iron-binding protein lactoferrin is a multifunctional protein that has antibacterial, antifungal, antiviral, antitumour, anti-inflammatory, and immunoregulatory properties. All of these additional properties appear to be related to its highly basic N-terminal region. This part of the protein can be released in the stomach by pepsin cleavage at acid pH. The 25-residue antimicrobial peptide that is released is called lactoferricin. In this work, we review our knowledge about the structure of the peptide and attempt to relate this to its many functions. Microcalorimetry and fluorescence spectroscopy data regarding the interaction of the peptide with model membranes show that binding to net negatively charged bacterial and cancer cell membranes is preferred over neutral eukaryotic membranes. Binding of the peptide destabilizes the regular membrane bilayer structure. Residues that are of particular importance for the activity of lactoferricin are tryptophan and arginine. These two amino acids are also prevalent in "penetratins", which are regions of proteins or synthetic peptides that can spontaneously cross membranes and in short hexapeptide antimicrobial peptides derived through combinatorial chemistry. While the antimicrobial, antifungal, antitumour, and antiviral properties of lactoferricin can be related to the Trp/Arg-rich portion of the peptide, we suggest that the anti-inflammatory and immunomodulating properties are more related to a positively charged region of the molecule, which, like the alpha- and beta-defensins, may act as a chemokine. Few small peptides are involved in as wide a range of host defense functions as bovine and human lactoferricin.     

Borate esters of the methyl D-glucopyranosides

Methyl alpha- and beta-D-glucopyranoside act as moderate chelators of a boron centre through their O-4/6 binding site whereas the trans/trans arrangement of the O-2/3/4 hydroxy functions is not suited to form a chelate with the small boron central atom. In this work we report the crystal structure of the bisdiolatoborate Na2[B(Me alpha-D-Glcp4,6H(-2))(Me alpha-D-Glcp3,4,6H(-3))].NaOH.8H2O (1) along with a combined 11B and 13C NMR spectroscopic studies of borate-pyranoside solutions at different concentrations and pH. It is shown that crystallisation of a bisdiolatoborate needs both a high pH and a high total concentration.     

Selective determination of arginine-containing and tyrosine-containing peptides using capillary electrophoresis and laser-induced fluorescence detection.

The use of two different amino acid-selective fluorogenic reagents for the derivatization of peptides is investigated. One such scheme utilizes a selective reaction of benzoin with the guanidine moiety to derivatize arginine residues occurring in a peptide. The second scheme involves the formylation of tyrosine, followed by reaction with 4-methoxy-1,2-phenylenediamine. The use of capillary electrophoresis and laser-induced fluorescence detection allows enhanced efficiencies and sensitivities to be obtained for the separations of either arginine- or tyrosine-containing peptides. A helium-cadmium laser (325 nm) is ideally suited for the laser-based detection system due to a close match of the excitation maxima of derivatized peptides from both reactions. A detection limit of 270 amol is achieved for model arginine-containing peptides, while the detection limit for model tyrosine-containing peptides is measured at 390 amol. Both derivatization reactions are found to be useful for high-sensitivity peptide mapping applications in which only the peptides containing the derivatized amino acids are detected.     

Antithrombin III regulates complement activity in vitro

Heparin, a polyion, exerts its main activity to inhibit coagulation through a serine protease inhibitor, antithrombin III. Previous studies have clearly shown that heparin in the absence of antithrombin III also has the capacity to regulate C activity. The present studies examined the ability of purified human antithrombin III to regulate classical and alternative pathways of C, alone and in the presence of heparin. Antithrombin III alone inhibited generation of both pathways in a dose-related manner; antithrombin III at 8 micrograms/10(7) cellular intermediates inhibited generation of the classical and alternative pathway convertases by 60 and 42%, respectively. Antithrombin III and heparin augmented each other's capacity to inhibit generation of both convertases in a dose-related manner. Antithrombin III did not appear to inhibit on the basis of charge because it is only slightly anionic (isoelectric pH value, 5.0); instead, antithrombin III may have acted as a serine protease inhibitor of the proteolytic enzymes of the C cascades. Antithrombin III acted only to inhibit formation of the alternative pathway convertase but had no activity on terminal lysis by this pathway; similarly, antithrombin III inhibited preformed EAC1,4b,2a,3b but had no activity on classical pathway cellular intermediates containing additional components. Finally, antithrombin III inhibited consumption of factor B hemolytic activity in a reaction mixture that also contained factor D and C3b, suggesting that factor D activity was also inhibited. These studies demonstrate the capacity of antithrombin III to regulate C and suggest that, in concert with heparin, antithrombin III may play an important role in the regulation of C in vivo.