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PubMed1998

[Isolation of trypsin PC from the Kamchatka crab Paralithodes camtschatica and its properties].

Trypsin PC from the hepatopancreas of the king crab Paralithodes camtschatica was isolated and purified to apparent homogeneity by ion-exchange chromatography on Aminosilochrom and DEAE-Sephadex and affinity chromatography on arginine-agarose. The yield of the enzyme was 37.7%, and the purification degree was 21. Trypsin PC has a molecular mass of 29 kDa and pI < 2.5. It hydrolysis N-benzoyl-L-arginine p-nitroanilide at the optimum pH of 7.5-8.0 and at the temperature optimum of 55 degrees C (K(m) = 0.05 mM). Trypsin PC retained its activity within the pH range of 5.8-9.0 in the presence of Ca2+. The enzyme was inhibited by the specific inhibitors of serine proteases diisopropyl fluorophoshate and phenylmethylsulfonyl fluoride, by the trypsin inhibitor N-tosyl-L-lysylchloromethylketone, and by the trypsin inhibitors from soybean and potato. Trypsin PC was found to hydrolyze amide bonds formed by carboxylic groups of lysine and arginine in peptide substrates. The N-terminal sequence of this enzyme is IVGGTEVTPG.

Amino Acid Sequence↗
PubMed2001

Effect of LY287045, a thrombin/trypsin inhibitor, on thrombin and trypsin-induced aortic contraction and relaxation.

The active site tripeptide arginal inhibitor of thrombin, LY287045, was used to study thrombin-induced aortic relaxation and contraction, two responses that differ both pharmacologically and physiologically. Although thrombin (10(-7) M) and trypsin (10(-6) M) were tachyphylactic upon repeated administration, trypsin contracted the aorta following thrombin-induced contraction. LY287045 (10(-7) M) attenuated thrombin-induced vasorelaxation, but not vasoconstriction with -log K(B) of 8.4. LY287045 (10(-7) M) also attenuated vasorelaxation, but not vasoconstriction to trypsin, another serine-protease with a thrombin-like catalytic triad, with similar potency (-log K(B) = 8.6) to that for thrombin. Consistent with these vascular effects, LY287045 inhibited the protease activity of both thrombin and trypsin. To explore further the selective inhibitory effect of LY287045 on protease-induced relaxation, we examined the effect of LY287045 on the nitric oxide and prostacyclin pathways and found that LY287045 did not alter vascular responses mediated by nitric oxide or prostacyclin. Likewise, LY287045 did not exert a direct inhibitory effect on the relaxant protease-activated receptor (PAR) since relaxation to the PAR-2-activating peptide was not blocked. The selective effect of LY287045 to inhibit only protease-induced endothelial-dependent relaxation demonstrated that protease inhibition will not affect all protease responses equally. Furthermore, increases in trypsin and thrombin have been associated with inflammation and angiogenesis. To the extent that these findings suggest that LY287045 exhibit dual protease inhibition of endothelial responses, LY287045 may have specific utility in hypotensive inflammatory diseases and in cancer metastases where both trypsin and thrombin have been implicated as causative agents.

Animals↗
PubMed2001

[Biological evaluation of soybean line with low trypsin inhibitor activities].

The soybean cultivar BR 36 with conventional levels of trypsin inhibitors activity and the soybean line BRM95-5262, which was genetically selected to contain low activity of trypsin inhibitors were used for biological assays with rats. BR 36 and BRM95-5262 contained 40 and 20, and 30 and 20% of relative residual activity of trypsin inhibitors, respectively. The mean values of PER and NPR showed that treatments with crude soybeans were minor than treatments with soybean thermically processed. However, the treatments of thermically processed soybean did not showed significative differences (p > or = 0.05). When the trypsin inhibitors activity were 8.61 and 8.44 UIT/mg of samples or 20 and 30% of relative residual activity of cultivar BR 36 and line BRM95-5262, respectively, it was observed that mean values of PER and NPR were not significatives. The mean values of CDA and CDV of treatments with crude soybeans were minor than treatment with casein and similar to the treatments with soybean thermically processed. So, it can be concluded that the biological evaluation obtained with soybean protein were dependent of initial trypsin inhibitors activities and of its respective thermical treatment. There was advantage in the use of BRM95-5262 soybean line with low trypsin inhibitors activity.

Animals↗
PubMedDate not supplied

[Effect of the degree of modification of trypsin amino groups with polymers on the enzymatic and conformation properties of the protein].

Trypsin was modified by introducing fragments containing an azo-bond into its molecule by the reaction of free amino groups of the enzyme with an azide of 2,2'-azobisisobutryic acid. Subsequently free-radical polymerization of N-vinyl pyrrolidone was carried out with the high molecular weight initiator obtained. The degree of modification of amino groups in trypsin was n = 6 divided by 12, which distinguishes this type of modification from that earlier proposed by the authors. In that case dichlorohydrate of dimethylimidate of 2,2'-azobisisobutyric acid was used for introducing azo-bonds into the molecule of the protein, n being equal to 2-3. It is shown that under the conditions of autolytic degradation both high molecular weight initiator based on trypsin and the trypsin-PVP (poly-N-vinyl pyrrolydone) covalent conjugates exhibit higher stability than initial trypsin. The method of circular dichroism was used for comparison of conformational properties of the modified trypsin forms. An increase of the rate of thermal inactivation was found to result from conformational changes occurring on modification of the enzyme.

Amines↗
PubMed1981

Trypsin inactivation by intact Hymenolepis diminuta (Cestoda): some characteristics of the inactivated enzyme.

In the presence of intact Hymenolepis diminuta, trypsin was inactivated; intact worms had no apparent effect on subtilisin, pepsin, or papain. Inactivation of trypsin was demonstrable using azoalbumin as a substrate, but the inactivated enzyme retained full catalytic activity against benzoyl-DL-arginine-p-nitroanilide, p-tosyl-L-arginine methyl ester (low molecular weight synthetic trypsin substrates) and p-nitro-p-guanidinobenzoate (an active site titrant). Inactivation was not reversible under conditions of heating, freezing and thawing, or prolonged dialysis of the enzyme. Analyses of inactivated 3H-trypsin by cationic and SDS-polyacrylamide gel electrophoresis, and gel chromatography failed to indicate the presence of a high molecular weight trypsin inhibitor associated with the inactivated enzyme; no low molecular weight, dissociable inhibitor was demonstrable following thermal denaturation of the inactivated enzyme. Analyses of trypsin after incubation in the presence of pulse-labeled worms also failed to demonstrate the presence of any inhibitor of worm origin associated with the inactivated enzyme. The data suggest that inactivation is the result of a small structural or conformational change in the enzyme molecule, a change which partially (rather than totally) inactivates the enzyme towards protein substrates.

Albumins↗
PubMed1981

Studies on the influence of heparin on trypsin-induced shock in dog.

Intravenous infusion of bovine trypsin in dogs can induce intravascular coagulation, which is claimed to be one of the causes of the shock accompanying such infusions. Treatment with heparin prior to trypsin administration prevents the usual decrease of fibrinogen but apparently does not influence the trypsin-induced shock seen upon trypsin saturation of alpha 2-macroglobulin. The prompt appearance of fibrinogen fragments following trypsin saturation of alpha 2-macroglobulin is probably a consequence of plasminogen activation. Under similar conditions alpha 1-antitrypsin (alpha 1-protease inhibitor) seems to be acting as plasma inhibitor in the absence of free alpha 2-macroglobulin and the fast reacting plasmin inhibitor, antiplasmin. Thus, trypsin-induced shock in dogs is apparently not caused by intravascular coagulation to any significant degree.

Animals↗
PubMedDate not supplied

[Comparative study of active site structure in bovine and Pacific salmon trypsins].

The kinetic investigation was carried out on the inhibition of hydrolysis of N, alpha-benzoyl-D, L-arginine-p-nitroanilide (BApNA) for bovine and salmon trypsin by phenylmethanesulphonyl fluoride (PMSF), N, alpha-tosyl-L-lysine chloromethyl ketone (N-TLCK), N, alpha-tosyl-L-phenylalanine chloromethyl ketone (N-TPCK). Kinetic parameters of inhibition (Ki, k2) by PMSF for salmon and bovine trypsin differ insignificantly. The k2/Ki value of N-TPCK for salmon trypsin is 10 times more than of bovine trypsin. Kinetic parameters of inhibition by N-TLCK had the less difference. The Ki value of this inhibitor for salmon trypsin is 5 times less than that of bovine trypsin and k2 value is 1.7 times less.

Animals↗
PubMed2000

Kinetics of beta-casein hydrolysis by wild-type and engineered trypsin.

Apparent rate constants of tryptic hydrolysis of amide bonds containing Arg and Lys residues in beta-casein were determined by the analysis of kinetics of accumulation of 17 major peptide components revealed by high performance liquid chromatography. When studying pH influence on Arg/Lys bond cleavage preference, averaged rate constants over several Arg&bond;X and Lys&bond;X bonds were used for analysis of kinetics of wild-type trypsin, K188H, K188F, K188Y, K188W, and of K188D/D189K mutants. The pK(a1) value of 6.5 was found for all studied trypsins. For wild-type trypsin and its K188D/D189K mutant, pK(a2) was found to be 10. The lowest among studied engineered trypsins pK(a2) = 9.3 was determined for K188Y mutant. Considerable preference for the cleavage of Arg over Lys containing peptide bonds was demonstrated for all trypsins with engineered S2 site except for K188H and K188F. The comparison of individual rate constants for various bonds showed that during the hydrolysis by wild-type trypsin, the probabilities of splitting depend on secondary specificity and local hydrophobicity of amino acid residues, which are nearest to the hydrolyzed peptide bond (P2 site). The improvement of prediction of hydrolysis rates performed by the used program was achieved after considering the presence of hydrophobic neighborhood of Lys48--Ile49 and Arg202--Gly203 bonds.

Base Sequence↗
PubMed2003

Trypsin activity assay in substrate-specific one- and two-dimensional gels: a powerful method to separate and characterize novel proteases in active form in biological samples.

To separate and identify the proteases, a substrate-specific, sensitive assay in sodium dodecyl sulfate (SDS)-polyacrylamide gels after two-dimensional (2-D) electrophoresis has been developed. This method allows simultaneous determination of protease cleavage specificity, molecular weight, isoelectric point, and if necessary, amino acid sequencing. After isoelectric focusing in immobilized pH gradient (IPG) strips (pH 6-11) (first dimension), trypsin was electrophoresed in 12% SDS polyacrylamide gels (second dimension) copolymerized with Boc-Gln-Ala-Arg-MCA (4-methyl-coumaryl-7-amide). The gels were washed in cold 2.5% Triton X-100 and water, and incubated in assay buffer (6.3 mM Bicine, 100 mM NaCl). Trypsin cleavage of the peptide-MCA generated fluorescent 7-amino-4-methyl-coumarin. In 1-D gels, as low as 500 pg trypsin could be detected and trypsin band volumes correlated linearly with the amounts of trypsin (R(2) = 0.999). In 2-D gels, the lowest amount of trypsin detected was 1 ng. The linear regression of spot volume and loading amount was still good (R(2) = 0.974). To optimize renaturation conditions, 5x5 min washes with 2.5% Triton X-100 and water, respectively, gave the strongest band volume. For fluorescence development, an assay buffer at pH 9 was the best; incubation at 37 degrees C for 30 min was sufficient. The method has application for identifying novel proteases as it does not rely on antibodies.

Biological Assay↗
PubMed1994

Macromolecular chelation as an improved mechanism of protease inhibition: structure of the ecotin-trypsin complex.

The 2.4 A crystal structure (R = 0.180) of the serine protease inhibitor ecotin was determined in a complex with trypsin. Ecotin's dimer structure provides a second discrete and distal binding site for trypsin and, as shown by modelling experiments, other serine proteases. The second site is approximately 45 A from the reactive/active site of the complex and features 13 hydrogen bonds, including six that involve carbonyl oxygen atoms and four bridged by water molecules. Contacts ecotin makes with trypsin's active site are similar to, though more extensive than, those found between trypsin and basic pancreatic trypsin inhibitor. The side chain of ecotin Met84 is found in the substrate binding pocket of trypsin where it makes few contacts, but also does not disrupt the solvent structure or cause misalignment of the scissile bond. This first case of protein dimerization being used to augment binding energy and allow chelation of a target protein provides a new model for protein-protein interactions and for protease inhibition.

Bacterial Proteins↗
PubMed1994

Isolation of porcine pancreatic islets: low trypsin activity during the isolation procedure guarantees reproducible high islet yields.

During the past few years, interest in xenotransplantation of porcine islets of Langerhans for the future therapy of type I diabetes has increased markedly. Therefore, we established a semiautomated digestion method for isolating islets from the porcine pancreas. However, although the isolation technique was standardized and collagenase of controlled quality was used, we were unable to attain high islet yields with a satisfactory degree of reproducibility. One hypothesis was that varying degrees of interference by donor pancreatic enzymes were responsible for this failure. The aim of this study was to examine the kinetics of four types of enzymatic activity during the isolation procedure, as well as their effects on islet yield: collagenase, trypsin, neutral protease, and clostripain. Our results indicate that while exogenous collagenase activity decreases slightly during the isolation procedure, the activity of the pancreas enzymes neutral protease and trypsin increases. In some cases, trypsin activity increases very strongly. A strong increase in trypsin activity correlates with poor islet yield, whereas low trypsin activity always correlates with high islet yield. Addition of the protease inhibitor Pefabloc to the isolation medium results in low trypsin activity and reproducible high islet yields.

Animals↗
PubMed1975

Effect of cell trypsinization on nuclear proteins of WI-38 fibroblasts in culture.

When resting confluent monolayers of WI-38 fibrolasts are trypsinized and replated at a lower density they are stimulated to proliferate again with an interval of 18 hours between replating and the onset of DNA synthesis. Trypsinization of resting cells causes a 40% loss of nuclear proteins as well as of cytoplasmic proteins. The amount of nuclear proteins remains low for the first six hours after the cells have been replated and then it increases rapidly, reaching the same level of non-trypsinized resting cells by ten hours after plating. The proteins that are lost from the nucleus immediately after trypsinization are chromatin-associated proteins and most of them are non-histone chromosomal proteins, although a modest loss of histones cannot be ruled out. The loss of non-histone chromosomal proteins from cells that have been trypsinized causes changes in the structure of chromatin that can be detected by circular dichroism and by viscosity measurements. These results show that cell trypsinization causes an extensive loss of proteins from chromatin and that the loss is restored only several hours after the cells have been replated at a lower density.

Cell Line↗
PubMed1989

Capacitation of bovine spermatozoa by lysophospholipids and trypsin.

Bovine spermatozoa were incubated in vitro with lysophosphatidylserine (LPS), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI), or trypsin. Capacitation of sperm was evaluated by penetration of the zonae pellucidae of dead bovine oocytes. Capacitation times could be shortened to 3 h or less by treatment of spermatozoa with each of these lysophospholipids (LPLs) (P less than .05). The maximum oocyte penetration percentages for individual LPLs were 40% for 10 microM LPS. 24% for 160 microM LPC, 31% for 320 microM LPE, and 19% for 320 microM LPI. Capacitation also was facilitated (P less than .01) by trypsin treatment of spermatozoa. Spermatozoa treated with 250 or 2,500 units/ml of trypsin penetrated more oocytes (17 and 18%) than spermatozoa treated with 0 or 25 units/ml of trypsin (0 and 3%). Spermatozoa treated with increasing concentrations of LPL showed a decrease in both the percentage of intact acrosomes and of progressively motile spermatozoa. Increasing levels of trypsin in the incubation medium also led to a decrease (P less than .05) in the percentages of intact acrosomes and a decrease (P less than .01) in the percentages of progressively motile spermatozoa. Percentages of live, ovulated oocytes fertilized by spermatozoa incubated for 1 h in LPS (86%, 6/7) were not different from those incubated for 24 h in control medium (71%, 5/7). Percentages of oocytes fertilized with both of these capacitation treatments were higher (P less than .05) than for oocytes exposed or killed or uncapacitated sperm. Rapid induction of capacitation and the acrosome reaction can be accomplished by exogenous treatment of bovine sperm with lysophospholipids or trypsin.

Animals↗
PubMed1998

Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificity.

Cluster analysis is presented as a technique for analyzing the conservation and chemistry of water sites from independent protein structures, and applied to thrombin, trypsin, and bovine pancreatic trypsin inhibitor (BPTI) to locate shared water sites, as well as those contributing to specificity. When several protein structures are superimposed, complete linkage cluster analysis provides an objective technique for resolving the continuum of overlaps between water sites into a set of maximally dense microclusters of overlapping water molecules, and also avoids reliance on any one structure as a reference. Water sites were clustered for ten superimposed thrombin structures, three trypsin structures, and four BPTI structures. For thrombin, 19% of the 708 microclusters, representing unique water sites, contained water molecules from at least half of the structures, and 4% contained waters from all 10. For trypsin, 77% of the 106 microclusters contained water sites from at least half of the structures, and 57% contained waters from all three. Water site conservation correlated with several environmental features: highly conserved microclusters generally had more protein atom neighbors, were in a more hydrophilic environment, made more hydrogen bonds to the protein, and were less mobile. There were significant overlaps between thrombin and trypsin conserved water sites, which did not localize to their similar active sites, but were concentrated in buried regions including the solvent channel surrounding the Na+ site in thrombin, which is associated with ligand selectivity. Cluster analysis also identified water sites conserved in thrombin but not trypsin, and vice versa, providing a list of water sites that may contribute to ligand discrimination. Thus, in addition to facilitating the analysis of water sites from multiple structures, cluster analysis provides a useful tool for distinguishing between conserved features within a protein family and those conferring specificity.

Animals↗
PubMed1999

Properties of the His57-Asp102 dyad of rat trypsin D189S in the zymogen, activated enzyme, and alpha1-proteinase inhibitor complexed forms.

Structural and biochemical studies suggest that serpins induce structural rearrangements in their target serine-proteinases. Previous NMR studies of the complex between a serpin, alpha1-proteinase inhibitor, and a mutant of recombinant rat trypsin (the Asp189 to Ser mutant, D189S, which is much more stable than wild-type rat trypsin against autoproteolysis) provided information about the state of catalytic residues in this complex: the hydrogen bond between Asp102 and His57 remains intact in the complex, and spectral properties of His57 are more like those of the zymogen than of the activated enzyme (G. Kaslik, et al., 1997, Biochemistry 36, 5455-5464). Here we report the protonation and exchange behavior of His57 of recombinant rat trypsin D189S in three states: the zymogen, the active enzyme, and the complex with human alpha1-proteinase inhibitor and compare these with analogous behavior of His57 of bovine chymotrypsinogen and alpha-chymotrypsin. In these studies the pKa of His57 has been determined from the pH dependence of the 1H NMR signal from the Hdelta1 proton of histidine in the Asp102-His57 dyad, and a measure of the accessibility of this part of the active site has been obtained from the rate of appearance of this signal following its selective saturation. The activation of rat trypsinogen D189S (zymogen, pKa = 7.8 +/- 0.1; Hill coefficient = 0. 86 +/- 0.05) decreased the pKa of His57 by 1.1 unit and made the protonation process cooperative (active enzyme, pKa = 6.7 +/- 0.1; Hill coefficient = 1.37 +/- 0.08). The binding of alpha1-proteinase inhibitor to trypsin D189S led to an increase in the pKa value of His57 to a value higher than that of the zymogen and led to negative cooperativity in the protonation process (complex, pKa = 8.1 +/- 0. 1; Hill coefficient = 0.70 +/- 0.08), as was observed for the zymogen. In spite of these differences in the pKa of His57 in the zymogen, active enzyme, and alpha1-proteinase inhibitor complex, the solvent exchange lifetime of the His57 Hdelta1 proton was the same, within experimental error, in all three states (lifetime = 2 to 12.5 ms). The linewidth of the 1H NMR signal from the Hdelta1 proton of His57 was relatively sharp, at temperatures between 5 and 20 degrees C at both low pH (5.2) and high pH (10.0), in spectra of bovine alpha-chymotrypsin, recombinant rat trypsin D189S, and the complex between rat trypsin D189S and human alpha1-proteinase inhibitor; however, in spectra of the complex between alpha-chymotrypsin and human alpha1-proteinase inhibitor, the peak was broader and could be well-resolved only at the lower temperature (5 degrees C).

Amino Acid Substitution↗
PubMed1996

Selection of trypsin inhibitors in phage peptide library.

The newly developed techniques of peptide libraries have become a conventional and efficient method in screening ligands of proteins of interest. We present here the successful results of selection of trypsin inhibitors in a phage hexapeptide library. After affinity selection and activity assay, peptide sequences, deduced from DNA sequencing of the phage peptides with the most striking trypsin activity, share some common features with trypsin inhibitors reported. All of the phage peptides selected out and those native and synthetic trypsin inhibitors reported are composed of three parts: (a) positively charged part (Arg, Lys or their analogs); (b) polar part that may form hydrogen bonds with Ser in the active site of trypsin; (c) hydrophobic part that interacts with the nonpolar region of trypsin active site.

Amino Acid Sequence↗
PubMed1998

Two mutations in rat trypsin confer resistance against autolysis.

Due to autodigestion the activity of dissolved trypsin successively decreases. Autolysis leads to proteolytic cleavages of some arginyl and lysyl peptide bonds of the trypsin structure. Three important autolysis sites have been reported for bovine trypsin: Lys61-Ser62, Arg117-Val118 and Lys145-Ser146. Out of these three sites only the first two exist in rat trypsin, an enzyme that has been the target of protein engineering for more than ten years. In this work Lys61 and Arg117 were replaced by Asn via site directed mutagenesis to transform the corresponding peptide bonds to trypsin resistant ones. Kinetic parameters of K61N, R117N and the double mutant K61N/R117N are practically identical with those of the wild-type enzyme. By contrast, the rate of autolysis of each singly-substituted species is substantially slower than with the parent trypsin. In particular, the double mutant shows dramatically increased stability against autolysis and decreased sensitivity to Ca2+. The process of autolysis has been followed by N-terminal sequence determination. We propose a model to explain why these two positions play a key role in autolysis and how Ca2+ can influence this process. In addition, our in vitro results strongly support the recently proposed model of human hereditary pancreatitis.

Amino Acid Sequence↗
PubMed1998

Anti-autolysis of trypsin by modification of autolytic site Arg117.

In order to improve the stability of trypsin, an approach to knock out the autolytic site has been carried out in this investigation. Compared with trypsins from other species, the autolytic site Arg117-Val118 of rat trypsin is the most interesting candidate to work on. The Arg117 residue was designed to be deleted or replaced by other amino acid residues to destroy the autolytic site. With DNA site-directed mutagenesis method, one deletion mutant and several replacement mutants were selected. After expression and purification, the kinetic and anti-autolytic properties of mutant trypsins were studied. No net charge difference of the trypsin molecules was observed by native PAGE analysis. Kinetic studies show that the activities of mutants vary from one another. R117L gives 32 times the activity of wild type trypsin while R117C has no detective activity. Among 8 selected mutants with characteristic properties, 7 of them give prolonged half life during anti-autolytic assay with the exception of R117M which is more sensitive to autolysis.

Amino Acid Substitution↗