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PubMed1991

The sensitivity of cell-associated dengue virus proteins to trypsin and the detection of trypsin-resistant fragments of the nonstructural glycoprotein NS1.

Extracts of Vero cells infected with dengue virus type 2 were digested by trypsin in the presence and absence of detergents. The experiments were designed to test the models proposed for flavivirus translation in which the glycoproteins prM, E, and NS1 are inserted into the endoplasmic reticulum of the cell, whereas certain other nonstructural proteins are not. Viral polypeptides were detected by the use of radiolabel, by immunoprecipitation, or by immunoblotting. The results obtained for NS3 and NS5 were as predicted by the models, with membranes providing no protection against digestion by trypsin. Similarly, the results obtained for prM and E were consistent with the models, with membranes protecting against proteolysis. Some molecules of NS1 were protected, while others were sensitive to proteolysis; novel trypsin-resistant fragments of 69,000, 60,000, and 50,000 Mr (all heat-labile), and of 37,000 and 24,000 Mr were detected following treatment of cell extracts with various combinations of trypsin, detergent, and reducing agent. Preliminary experiments suggested that these tryptic fragments are potentially useful in mapping the antigenic epitopes of NS1.

Animals↗

PubMed1996

Alanine point-mutations in the reactive region of bovine pancreatic trypsin inhibitor: effects on the kinetics and thermodynamics of binding to beta-trypsin and alpha-chymotrypsin.

In an effort to relate structural, kinetic, and thermodynamic features in a model macromolecular recognition process, the amino acid residues in the reactive surface of bovine pancreatic trypsin inhibitor (BPTI) and surrounding residues were substituted individually by alanine, and the effects of the point-mutations on the kinetics and thermodynamics of inhibition by BPTI toward trypsin and chymotrypsin were investigated. Fifteen alanine mutants were produced. The majority of the BPTI mutants exhibited a binding affinity similar to that of the wild-type protein. The exceptions were the primary specificity site (PI) mutant and those mutants that seem to have nonlocal perturbations of structure, as revealed by circular dichroism and thermostability measurements. The mutation at the P1 site caused a reduction in the binding free energy of 10 and 1.8 kcal mol-1 for trypsin and chymotrypsin, respectively. The losses in binding affinity were determined almost exclusively by an increase in the dissociation rate constant. However, the rate of association of the P1 mutant, Lys-15-Ala, with trypsin was also drastically reduced (> 200-fold). Calorimetric measurements of the heats of binding for the association of chymotrypsin with the wild-type inhibitor and its alanine mutants allowed determination of the relative contributions of the changes in enthalpy and entropy to the free energy of binding. Compensatory changes in the two parameters were observed in several cases, which were attributed to desolvation effects at the binding interface.

Alanine↗

PubMed1999

Crystal structure of the complex formed between bovine beta-trypsin and MCTI-A, a trypsin inhibitor of squash family, at 1.8-A resolution.

The stoichiometric complex formed between bovine beta-trypsin and Momordica charantia, Linn. Cucurbitaceae trypsin inhibitor A (MCTI-A) was crystallized and its X-ray crystal structure was refined to a final R value of 0.179 using data of 7.0- to 1.8-A resolution. Combination with results on the complex of MCTI-A with porcine trypsin gives the sequence of MCTI-A definitely, of which 13 residues are conserved compared with other squash family trypsin inhibitors. Its spatial structure and the conformation of its primary binding segment from Cys3I (P3) to Glu7I (P3'), which contains a reactive scissile bond Arg5I C-Ile6I N, were found to be very similar to the other squash family proteinase inhibitors.

Amino Acid Sequence↗

PubMed1988

A predicted tertiary structure of a thrombin inhibitor-trypsin complex explains the mechanisms of the selective inhibition of thrombin, factor Xa, plasmin, and trypsin.

The tertiary structure of a thrombin inhibitor-trypsin complex has been predicted by a molecular modelling considering the van der Waals interactions between the inhibitor and the enzyme. The selective inhibition of trypsin, thrombin, factor Xa, and plasmin exhibited by arginine and lysine derivatives has been clearly explained based on the predicted structure and the homology in the amino acid sequences of these enzymes. The differences in the amino acid sequences at the positions corresponding to Ile63, Leu99, and Ser190 of trypsin give each enzyme different binding affinities toward inhibitors and result in the selective inhibition. The X-ray analysis of the inhibitor-trypsin complex is in progress to prove the predicted structure.

Amino Acid Sequence↗

PubMed1980

A novel chiral microenvironmental probe at the active site of trypsin. Extrinsic cotton effects of acyl-trypsin possessing an enantiomeric pair of chromophores.

p-Amidinophenyl esters of an enantiomeric pair of N-(2,4-dinitrophenyl)alanine (N2Ph-Ala) were both efficiently hydrolyzed by trypsin. The acylation and deacylation rate constants for the D-isomer are 1/2.5 of those for the L-isomer. Slow rates of deacylation of the two substrates made it possible to prepare the pair of enantiomeric acyl-trypsins. Circular dichroic (CD) spectra of the purified acyl-trypsins revealed that the two extrinsic chromophores are somewhat differently oriented in the chiral environment of the active site, although both chromophores could couple intermolecularly with similar intrinsic chromophores near the active site. When p-amidinophenol was added, not only was the deacylation rate of N2Ph-DAla-trypsin noticeably increased, but also the transient CD spectrum of the enzyme derivative changed markedly in comparison with that of the L-derivative. The observations indicate that the two enantiomeric acyl groups at the active site are situated in different microenvironments.

Acylation↗

PubMed1991

Binding of viral glycoprotein with trypsin and its relation to virulency. II. Comparison between bovine and Streptomyces griseus trypsins.

The two step nature of the binding reaction between trypsin and viral glycoproteins was further investigated using two types of trypsin, bovine trypsin and Streptomyces griseus trypsin. The experimental results were explained by the van der Waals energy operating in the second step, suggesting that the conformational aspects, in addition to the electrostatic nature, of the interacting peptides are decisive in this specific process.

Amino Acid Sequence↗

PubMed1989

Kinetic study on the mechanism of inhibition of trypsin and trypsin-like enzymes by p-guanidinobenzoate ester.

It was found that [4-(2-succinimidoethylthio)phenyl 4-guanidinobenzoate]methanesulfonate (E-3123) inhibits trypsin, thrombin and kallikrein, and its inhibitory activity is most potent toward trypsin. The interactions of these enzymes with E-3123 were studied mainly by using stopped-flow spectrophotometry. E-3123 behaved as a quasi-substrate of the enzymes and the inhibitory property was due to the efficient production of the stable acyl-enzyme. The acylation process with trypsin was exceedingly effective, and the resulting acyl-enzyme was the most stable among the three enzymes tested. This observation affords a rational basis for explaining the action of E-3123, which is a transient inhibitor most active toward trypsin.

Guanidines↗

PubMed1984

Discrepancy between the potency of various trypsin inhibitors to inhibit trypsin activity and the potency to release biologically active cholecystokinin-pancreozymin.

Injection of various trypsin inhibitors into the lumen of the isolated perfused rat duodenum increased the amount of biologically active cholecystokinin-pancreozymin (CCK-BA) in the vascular perfusate. The potency to induce CCK-BA release of the various trypsin inhibitors differed. Injection of ethyl p-(6-guanidinohexanoyloxy) benzoate methanesulfonate (FOY-007; 100 mumol), p-ethoxy-carbamoyl-thio-6-guanidino caproate phosphate (FOY-129; 110 mumol), 4-(4-guanidino-benzoyloxy) phenylacetic acid (FOY-251; 128 mumol), N,N-dimethyl-carbamoylmethyl-4-(4-guanidinobenzoyloxy) phenylacetate methanesulfonate (FOY-305; 80 mumol), and p-aminobenzamidine dihydrochloride (p-ABA, Sigma; 300 mumol) caused release of CCK-BA amounting to 1,042, 247, 252, 682, and 302 pM, respectively. The potency to induce CCK-BA release was not correlated with the potency to inhibit trypsin activity. The present results do not support the hypothesis that a negative feed-back regulation of pancreatic enzyme secretion is exerted by intraluminal trypsin in the rat.

Animals↗

PubMed1981

[Mechanism of thermal denaturation of native trypsin and trypsin modified by neutral soluble polymer].

The thermal denaturation of trypsin solutions and trypsin vinylpyrrolidone-acrolein copolymer conjugate was studied within the temperature range of 37.5-96 degree. pH 4.5. A mechanism for trypsin thermal denaturation based on its ability to form dimers and determining the minimal stability of the enzyme at 66 degree and its maximal stability at 74 degree is proposed. The thermal denaturation of the trypsin conjugate is of a different type, which depends on the changes in the thermal denaturation mechanism due to its incapability to form dimers. A decrease in the activation and thermodynamic parameters of the enzyme denaturation during its modification is due to the approximation of the protein structure to that of the denatured form, eventually resulting in the enzyme destabilization. The increased stability of the modified protein to the denaturating effects of low temperatures is due to the changes in the denaturation mechanism induced by enzyme modification.

Acrolein↗

PubMed1980

Radioimmunoassay for human pancreatic secretory trypsin inhibitor: measurement of serum pancreatic secretory trypsin inhibitor in normal subjects and subjects with pancreatic diseases.

A reliable radioimmunoassay (RIA) for human pancreatic secretory trypsin inhibitor (PSTI) has been developed. The method is highly sensitive (0.4 ng/ml), reproducible and specific. A good parallel relationship was observed between the standard curve and dilution curves for serum and urine. The PSTI bound to trypsin-alpha 2-macroglobulin complexes was found not to be immunoreactive, whereas a part of the psti-trypsin complex was immuno-reactive. In healthy individuals, serum PSTI level ranged from 5.4 ng/ml to 16.0 ng/ml, the average being 11.3 ng/ml (S.D. +/- 2.7). Elevated values were observed in patients with acute pancreatitis (highest value 3200 ng/ml), and in some patients with chronic relapsing pancreatitis.

Acute Disease↗

PubMed1993

High-performance capillary electrophoresis for the determination of trypsin and chymotrypsin inhibitors and their association with trypsin, chymotrypsin and monoclonal antibodies.

High-performance capillary electrophoresis (HPCE) was adapted for the determination of Kunitz soybean trypsin inhibitor, Bowman Birk inhibitor from soybean and protein-type proteinase inhibitors from pea (Pisum sativum L.). The method was developed for the determination and characterization of the inhibitors, the enzymes trypsin and chymotrypsin and the monoclonal antibodies (mAbs) raised against the inhibitors, and also the inhibitor-enzyme and inhibitor-mAb association complexes. The results from studies involving the use of various types of buffers revealed the advantages of having zwitterions such as trimethylammoniumpropyl sulphonate (AccuPure) or taurine included in the buffer. The use of capillaries dynamically coated with zwitterions efficiently reduced the interactions of the proteins with the silica capillary surface, which was important for the analyses for trypsin, chymotrypsin and mAbs and their association complexes with the inhibitors. The influence of temperature, voltage, pH and buffer type on migration times, resolution, peak areas and number of theoretical plates was investigated for the proteins studied. The proposed HPCE method is very suitable for studies of proteinase inhibitors compared with traditional inhibitor studies, and it gives efficient protein separations with the possibility of 245,000 plates/m.

1-Propanol↗

PubMed1975

Hydrogen exchange kinetics changes upon formation of the soybean trypsin inhibitor-trypsin complex.

The hydrogen exchange kinetics of the complex of trypsin-soybean trypsin inhibitor (Kunitz) have been compared to the calculated sum of the exchange kinetics for the inhibitor and trypsin measured separately. The exchange rates observed for the complex are substantially less than the sum of the exchange rates in the two individual proteins. These results cannot be accounted for by changes in intermolecular or intramolecular hydrogen bonding. The decrease in exchange rates in the complex are ascribed to changes in solvent accessibility in the component proteins.

Benzamidines↗

PubMed1987

Purification and sequencing of a trypsin-sensitive cholecystokinin-releasing peptide from rat pancreatic juice. Its homology with pancreatic secretory trypsin inhibitor.

The trypsin-sensitive cholecystokinin-releasing peptide is a peptide purified from rat pancreatic juice on the basis of its stimulatory activity toward pancreatic enzyme secretion. We postulate that the peptide acts as a mediator of pancreatic enzyme secretion in response to dietary protein intake and that it (designated as "monitor peptide" from its role in the intestine) could be responsible for the feedback regulation of pancreatic enzyme secretion. About 20 nmol of the highly purified peptide were obtained from 800 ml of rat pancreatic juice by reverse-phase high performance liquid chromatography. It was then sequenced. The peptide comprises 61 amino acid residues (Table I). It has a sequence that closely resembles that of a highly conserved region in pancreatic secretory trypsin inhibitors (PSTIs, Kazal type inhibitor): -Ile-Tyr-Asx-Pro-Val-Cys-Gly-Thr-Asx-Gly-. However, the peptide is less related to other mammalian PSTIs than they are to each other. The additional 5 residues at the NH2 terminus make the peptide larger than the common 56-residue PSTIs. The trypsin-sensitive cholecystokinin-releasing peptide is to be classified as a Kazal-type inhibitor and may be one of the rat PSTIs or a related peptide. The present results and increasing evidence from other laboratories and ours suggest that Kazal-type inhibitors play previously unrecognized multiple physiological roles.

Amino Acid Sequence↗

PubMedDate not supplied

[Conformational aspects of beta-trypsin interactions with substrates and pancreatic trypsin inhibitor. II. Structure of tetrahedral adducts and acylenzyme].

Theoretical conformational analysis of the tetrahedral complexes of trypsin with the N-acetyl-L-lysine methyl amide, which are formed at the acylation and the deacylation stages of the catalytical act has been carried out. The lowest energy conformations are shown to be productive ones. All favorable structures of N-acetyl-L-lysyl-trypsin and N-acetyl-L-arginyl-trypsin acylenzymes have been analysed. The global conformations of both complexes are found to be very similar with the structures providing for a transition to the second tetrahedral state. Conformations of the nonbonded, tetrahedral and acyl complexes with N-acetyl-L-lysine methyl amide are compared and the differences in orientation of atomic groups participating in the catalysis are revealed. All changes of optimal structures of the complexes indispensable for the catalytical process are shown to proceed in a spontaneous way without introduction of any intramolecular strain.

Acetylation↗

PubMed1998

Kunitz-type soybean trypsin inhibitor revisited: refined structure of its complex with porcine trypsin reveals an insight into the interaction between a homologous inhibitor from Erythrina caffra and tissue-type plasminogen activator.

The Kunitz-type trypsin inhibitor from soybean (STI) consists of 181 amino acid residues with two disulfide bridges. Its crystal structures have been determined in complex with porcine pancreatic trypsin in two crystal forms (an orthorhombic form at 1.75 A resolution and a tetragonal form at 1.9 A) and in the free state at 2.3 A resolution. They have been refined to crystallographic R-values of 18.9%, 21.6% and 19.8%, respectively. The three models of STI reported here represent a significant improvement over the partial inhibitor structure in the complex, which was previously determined at a nominal resolution of 2.6 A by the multiple isomorphous replacement method. This study provides the first high-resolution picture of the complex between a Kunitz-type proteinase inhibitor with its cognate proteinase. Many of the external loops of STI show high B-factors, both in the free and the complexed states, except the reactive site loop whose B-factors are dramatically reduced upon complexation. The reactive site loop of STI adopts a canonical conformation similar to those in other substrate-like inhibitors. The P1 carbonyl group displays no out-of-plane displacement and thus retains a nominal trigonal planar geometry. Modeling studies on the complex between a homologous Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra and the human tissue-type plasminogen activator reveal a new insight into the specific interactions which could play a crucial role in their binding.

Amino Acid Sequence↗

PubMed1984

On the potential role of trypsin and trypsin inhibitors in acute pancreatitis.

The protective role of alpha 2-macroglobulin, alpha 1-antitrypsin and Aprotinin against trypsin-induced effects on C3 and kininogen was studied in a human in vitro model. When human cationic trypsin was added to human serum or plasma, there was a gradual saturation of alpha 2-macroglobulin and later of alpha 1-antitrypsin. When alpha 2-macroglobulin was 70% saturated, there was a prompt cleavage of both C3 and kininogen, in spite of 80% free and active alpha 1-antitrypsin. These biochemical changes and antiprotease levels are identical to our findings in patients with acute pancreatitis, especially in their peritoneal exudate. Very high concentrations of Aprotinin, 5-15 times higher than ever used clinically, blocked the cleavage of both C3 and kininogen, while doses commonly used clinically were without significant effect. The clinical implications are: A trypsin-induced activation of both the complement and kinin system with clinical consequence is possible in patients with acute pancreatitis because of very low alpha 2-macroglobulin levels. Aprotinin in adequate doses, 5-15 times higher than ever used clinically, seems to protect against activation of two systems.

Acute Disease↗

PubMed1989

A radioactive method for the measurement of trypsin and trypsin-like activities.

A simple and highly sensitive method for the assay of trypsin has been developed by making use of the phosphorylated synthetic peptide Leu-Arg-Arg-Ala-Ser-(32P)-Leu-Gly as substrate. The technique has been adapted from the phosphocellulose method of R. Roskoski, Jr. (in Methods in Enzymology (Corbin, J., and Hardman, J., Eds.), Vol. 99, pp. 3-6, Academic Press, New York) used for measuring of protein kinases. In addition to measuring the activity of trypsin at the microgram level, the 32P-labeled peptide method can be used for measuring other trypsin-like enzymes. It has been successfully utilized for the identification of a new peptidase from the fungus Saccobolus platensis.

Adenosine Triphosphate↗

PubMed1995

A specific immunoradiometric assay of cationic trypsin(ogen) that does not recognize trypsin-alpha-1-proteinase inhibitor complex.

An immunoradiometric assay using two monoclonal antibodies directed to human trypsin 1 was developed for measuring trypsin(ogen) in biological fluids. The assay is different from other assays in that it is specific for cationic trypsinogen and does not recognize the alpha-1-proteinase inhibitor-trypsin complex. It can be used as a complement to classical immunoassays to characterize trypsinogen activation in pathological cases. The evaluation and the specificity of the assay are presented.

Alpha-Globulins↗