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PubMed1977

Effect of dietary raw soybean and soybean trypsin inhibitor on trypsin and chymotrypsin activities in the pancreas and in small intestinal juice of growing swine.

Sixty-eight growing gilts with a 12 kg average initial weight were used in seven trials to study the effect of dietary raw soybean (Harosov) and SBTI (Kunitz soybean trypsin inhibitor) on pancreatic and small intestinal trypsin and chymotrypsin activities. A solvent-extracted, heated soybean meal (SBM) was used, cause reduced growth. Both a single-meal and continuous feeding of the raw soybean diet caused a decrease in the pancreatic trypsin and chymotrypsin activities. In contrast, to the rat and the chick, the pancreas of the pigs did not enlarge subsequent to consumption of the raw soybean or SBTI diets. Raw soybean feeding also resulted in an inhibition of the intestinal trypsin and chymotrypsin activities. This inhibiting effect was greater than that of the SBTI, especially the chymotrypsin-inhibiting effect. This suggested that soybean constituents other than the SBTI, such as the Bowman-Birk inhibitor, caused inhibition. In the pig the inhibition of the intestinal proteolysis may be a major cause of reduced growth when raw soybean is fed.

Animals↗
PubMed1983

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, IX. Isolation and characterization of the inhibitory parts of inter-alpha-trypsin inhibitors from several mammalian sera.

The inhibitory parts of inter-alpha-trypsin inhibitor-like proteins from several mammalian sera (sheep, goat, horse, donkey, pig, rabbit, rat and dog) were released by limited proteolysis with trypsin and were isolated by reversible binding to immobilized trypsin. The inhibitors are very similar with respect to their stability in acids, molecular masses and amino-acid compositions. They are different, however, in their inhibitory properties. In view of the known covalent structures of the inhibitory parts of the human and bovine inhibitors, homologous covalent structures consisting of two tandem Kunitz-type domains are suggested also for the isolated inhibitors. Bovine trypsin, bovine chymotrypsin and porcine plasmin are inhibited by all investigated inhibitors, most likely via their C-terminal domain. The inhibitors from horse, donkey, rabbit, rat and dog serum interact also with elastase from human polymorphonuclear granulocytes, those from sheep, goat and pig serum inhibit in addition porcine pancreatic elastase and bovine chymotrypsin via their N-terminal Kunitz-type domain. It is supposed that the amino-acid residue in position P1 of the N-terminal Kunitz-type domain is responsible for the characteristic inhibitory properties of each inhibitor.

Alpha-Globulins↗
PubMed1975

The partial amino acid sequence of trypsin inhibitor II from garden bean, Phaseolus vulgaris, with location of the trypsin and elastase-reactive sites.

The amino acid sequences of garden bean (Phaseolus vulgaris) trypsin inhibitor II and a related molecular species II' have been examined. The entire sequence of II' has been determined with the exception of five internal residues. The garden bean inhibitors are highly homologous to the Bowman-Birk soybean inhibitor and lima bean trypsin inhibitor IV. The trypsin-reactive site has been located in the second half of the molecule, while the first reactive site has been found to be directed against elastase. Garden bean inhibitor II (and II') is thus a double-headed inhibitor, simultaneously inhibiting 1 molecule of trypsin and 1 of elastase.

Amino Acid Sequence↗
PubMed1987

Crystal structure determination of mung bean trypsin inhibitor Lys fragment-bovine trypsin complex--molecular replacement, electron density map at 3.0 angstron resolution.

The orientation and position of the trypsin molecule in the complex crystal cell mung bean trypsin inhibitor Lys fragment (MBILF)-bovine trypsin (BTRY) have been successfully determined by molecular replacement method with the model of the refined bovine trypsin molecule. Starting from the BTRY coordinates which were oriented and located in the correct azimuth and position in the complex cell according to the result from rotation function and translation function, sim-weighted Fourier map with coefficients 2/Fo/-/Fc/ at 3.0 A resolution was calculated. Besides the electron density which is obviously attributed to itself, in the vicinity of the active site of BTRY the dense contour levels corresponding to the MBILF and and its boundary could be clearly seen in this map. The size of MBILF was approximately estimated at 15 x 15 x 25 A.

Crystallography↗
PubMedDate not supplied

[Conformational aspects of beta-trypsin interaction with substrates and pancreatic trypsin inhibitor. I. Conformational properties of residues in the enzyme-active center and the structure of a non-valent enzyme-substrate complex].

The theoretical conformational analysis of potential surfaces of Ser-195, His-57, Asp-102 and Gln-192 side chains in the active center of native beta-trypsin has been carried out. The above residues are shown to exist in low-energy conformational states in the free enzyme and in its nonbonded substrate complexes. Interrelations between the flexibility of the residues and their catalytical functions were revealed. Conformational aspects of interaction of trypsin with N-acetyl-L-lysine and N-acetyl-L-alanyl--L-alanyl--L-lysyl--L-alanyl methyl amide and with the Gly-12--Ile-19 BPTI fragment were analysed. The productive binding of the substrate at the nonbonded complex stage is shown to take place exclusively in the lowest energy conformation of the enzyme-substrate complex. Basing on theoretical and experimental evidence, the problems of primary and secondary specificity of trypsin, and potential properties of the native protein to form a productive nonbonded complex and to react at the subsequent stages of the catalytical act are discussed. Conformational changes in the active center and interactions with a substrate are shown to result from stabilizing enzyme-substrate interactions. Trypsin inhibition by BPTI molecule does not take place at the nonbonded complex stage.

Amino Acid Sequence↗
PubMed1994

The preparation and use of biotinylated trypsin in western blotting for the detection of trypsin inhibitory proteins.

Methods were developed for the preparation of biotinylated trypsin of high specific activity. This was used as a probe for the detection of serine proteinase inhibitory proteins which had been separated by sodium dodecyl sulfate-polyacrylamide gradient slab gel electrophoresis and electroblotted to nitrocellulose. The method was extremely sensitive and specific and could detect 0.15 ng of the serine proteinase inhibitor, trasylol (0.023 pmol active inhibitor). The method was also widely applicable and could be used to detect a range of serine proteinase inhibitors in human serum with molecular weights of 50 to approximately 180 kDa, soybean trypsin inhibitor variants, and secretory leukocyte proteinase inhibitor extracted from human intervertebral disc and articular cartilage. The identity of several of the serine proteinase inhibitors detected using biotinylated trypsin was verified by Western blotting using specific antibodies. Under the conditions used, electrotransfer of trasylol was quantitative; densitometric examination of blots indicated that there was a linear relationship between the amount of active trasylol electrophoresed (1-50 ng) and the intensity of the blot obtained with biotinylated trypsin as probe, indicating that serine proteinase inhibitory proteins may also be quantified by this technique using densitometric scanning.

Animals↗
PubMed1997

Distance between the basic group of the amino acid residue's side chain in position P1 of trypsin inhibitor CMTI-III and Asp189 in the substrate pocket of trypsin has an essential influence on the inhibitory activity.

Three new analogues of trypsin inhibitor CMTI-III were synthesized by the solid-phase method: [Lys5]-CMTI-III, [Orn5]CMTI-III and [Dab5]CMTI-III. Only one analogue with L-lysine residue in position P1 showed inhibitory activity of the same order of magnitude as did wild CMTI-III. Two remaining analogues were completely inactive. A conclusion was drawn that the distance between the basic group of the amino acid residue's side chain in position P1 of the trypsin inhibitor CMTI-III and Asp189 in the substrate pocket of trypsin plays an essential role for the trypsin-inhibitor interaction.

Amino Acid Sequence↗
PubMed1989

Crystallization and preliminary X-ray study of porcine trypsin, free and complexed with Ecballium elaterium trypsin inhibitor, a member of the squash inhibitors family.

Porcine trypsin has been crystallized either free or complexed with synthetic Ecballium elaterium trypsin inhibitor II, a 28-residue peptide with three disulfide bridges. The crystals diffract beyond 2.0 A. Crystals are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 77.32 A, b = 53.81 A, c = 46.91 A, for the free trypsin, and a = 62.25 A, b = 62.27 A, c = 84.66 A for the complex with E. elaterium trypsin inhibitor II.

Animals↗
PubMed2005

Trypsin inhibition by a peptide hormone: crystal structure of trypsin-vasopressin complex.

The large variety of serine protease inhibitors, available from various sources such as tissues, microorganisms, plants, etc., play an important role in regulating the proteolytic enzymes. The analysis of protease-inhibitor complexes helps in understanding the mechanism of action, as well as in designing inhibitors. Vasopressin, an anti-diuretic nonapeptide hormone, is found to be an effective inhibitor of trypsin, with a K(i) value of 5 nM. The crystal structure of the trypsin-vasopressin complex revealed that vasopressin fulfils all the important interactions for an inhibitor, without any break in the scissile peptide bond. The cyclic nature due to a disulfide bridge between Cys1 and Cys6 of vasopressin provides structural rigidity to the peptide hormone. The trypsin-binding site is located at the C terminus, while the neurophysin-binding site is at the N terminus of vasopressin. This study will assist in designing new peptide inhibitors. This study suggests that vasopressin inhibition of trypsin may have unexplored biological implications.

Binding Sites↗
PubMed1968

The behaviour of trypsin towards alpha-N-methyl-alpha-N-toluene-p-sulfonyl-L-lysine beta-naphthyl ester. A new method for determining the absolute molarity of solutions of trypsin.

1. alpha-N-Methyl-alpha-N-toluene-p-sulphonyl-l-lysine beta-naphthyl ester (MTLNE) was synthesized as its hydrobromide and shown to be slowly hydrolysed by bovine pancreatic trypsin. The acylation step, however, is so much faster than deacylation of the acyl-enzyme that spectrophotometric measurement of the ;burst' of beta-naphthol provides a convenient method for determining the absolute molarity of trypsin solutions. 2. By using the same stock solution of trypsin, application of this method at pH4.0 and pH7.0 as well as that of Bender et al. (1966) at pH3.7 gave concordant results. 3. Provided that [S](0)>[E](0), the size of the ;burst' is independent of substrate concentration. 4. In the trypsin-catalysed hydrolysis of alpha-N-toluene-p-sulphonyl-l-arginine methyl ester, MTLNE functions as a powerful non-competitive inhibitor. 5. There is no detectable reaction between MTLNE and either bovine pancreatic alpha-chymotrypsin at pH4.0 or bovine thrombin at pH6.0.

Animals↗
PubMed1972

Trypsin action on the growth of Sendai virus in tissue culture cells. II. Restoration of the hemolytic activity if L cell-borne Sendai virus by trypsin.

Sendai virus grown in L cells (L Sendai) caused little hemolysis, whereas the one grown in fertile eggs (egg Sendai) induced distinct hemolysis. Enzymatic treatment with trypsin at low concentrations markedly enhanced the hemolytic activity of L Sendai but not that of egg Sendai. Both sonic treatment and freezing and thawing greatly enhanced the hemolytic activity of egg Sendai, but they gave little enhancing effect on that of L Sendai which could, however, be greatly increased by successive treatment with trypsin. Dose response and kinetic experiments on the trypsin effect have suggested that a similarity exists in the inhibitory mechanism of infectivity for L cells and hemolytic activity of L Sendai. Treatment of L cells with trypsin at later stages of infection released a highly hemolytic L Sendai from those cells. The present study, by reference to the density centrifugation studies in a previous report (4), has shown that a variation in infectivity for L cells and in the hemolytic activity of L Sendai is a type of host-controlled modification distinguishable from the density variation.

Animals↗
PubMed1981

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, IV. The amino acid sequence of the human urinary trypsin inhibitor isolated by affinity chromatography.

An acid-resistant trypsin inhibitor from human urine and serum is released in vivo by limited proteolysis from the high molecular acid-labile inter-alpha-trypsin inhibitor. The inhibitor shows an apparent molecular mass of 30 000 Da and is composed of two Kunitz-type domains. The domains are released in vitro by prolonged tryptic hydrolysis. The C-terminal domain is responsible for antitryptic activity. For the other domain no inhibitory activity towards proteinases, i.e. chymotrypsin, trypsin, pancreatic and leucocytic elastase has been demonstrated so far. The polypeptide chain comprising both domains consists of 122 residues and has a molecular mass of only 13 400 Da. In this work we have found that both, the N-terminal extension peptide with 21 residues and the "inactive" domain are linked O-glycosidically and N-glycosidically, respectively, with large carbohydrate moieties. The N-terminal amino acid sequence of the human urinary trypsin inhibitor was determined by solid-phase Edman degradation of a single peptide. The molecular mass calculated for the total polypeptide chain of 143 residues should be 15 340 Da; from the difference to the measured value (30 000 Da) it is concluded that the glycopeptide contains a considerable carbohydrate moiety.

Alpha-Globulins↗
PubMed1999

Kinetics of interaction of trypsin with an anionic inhibitor of trypsin BWI-1a from buckwheat seeds.

The kinetics of binding of bovine trypsin to a proteinaceous inhibitor of trypsin from buckwheat seeds (BWI-1a) has been studied. The association rate constant (k(ass)) was 2.2 x 10(6) M-1 x sec-1 and the dissociation rate constant (k(off)) of the enzyme--inhibitor complex was 3.5 x 10(-3) sec-1; the inhibition constant Ki was 1.5 nM. The inhibitor BWI-1a is of the slow, tightly binding type. The mechanism of the inhibition of bovine trypsin by the trypsin inhibitor BWI-1a was studied. The mechanism of inhibition was found to involve two steps according to the kinetic data.

Animals↗
PubMedDate not supplied

[Interaction of trypsin with a trypsin inhibitor from bovine colostrum].

Kinetics of trypsin association with trypsin inhibitor from colostrum (IC) was studied. The association rate constant is 3-10-5 M- minus 1 sec- minus 1 at pH 7,8, 25 degrees C. The rate constant for the complex dissociation was determined from the kinetics of the IC displacement from the complex with trypsin by a specific substrate and was found to be 5-10- minus 6 sec- minus 1 (pH 7,8; 25 degrees C). The equilibrium constant (Ki) was measured in a special experiment and was equal to 4-10- minus 12 M (p H 7,8; 25 degrees C). The similarity of this reaction and the association of trypsin with other protein inhibitors was discussed.

Animals↗
PubMed1982

Automated assay for alpha 1-antitrypsin with N-alpha-benzoyl-DL-arginine-p-nitroanilide as trypsin substrate and standardized with p-nitrophenyl-p'-guanidinobenzoate as titrant for trypsin active sites.

alpha 1-Antitrypsin is the most abundant of several serum protease inhibitors. Its deficiency is associated with an increased incidence of emphysema in adults, jaundice in newborns, and childhood cirrhosis. We describe an automated functional assay for the Instrumentation Laboratory's Multistat III Microcentrifugal Analyzer with N-alpha-benzoyl-DL-arginine-p-nitroanilide as trypsin substrate. The assay is standardized in terms of moles of trypsin active sites inhibited per liter of serum, by use of a chromogenic titrant for trypsin active sites, p-nitrophenyl-p'-guanidinobenzoate. The method is rapid, precise, and independent of trypsin supplier, and results correlate well with those by a manual chromogenic and a nephelometric assay.

Adult↗
PubMed2002

Trypsin-catalysed synthesis of oligopeptide amides: comparison of catalytic efficiency among trypsins of different origin (bovine, Streptomyces griseus and chum salmon).

A procedure has been developed for the synthesis of oligopeptide amide using inverse substrates as acyl donors with amino acid amide instead of p-nitroanilide as acyl acceptor and trypsins of different origin (bovine, Streptomyces griseus and chum salmon trypsins) as the catalyst. The effectiveness of this procedure was demonstrated by the synthesis of a pentapeptide, Boc-[Leu5]-enkephalin amide, as a model compound. The method was the first enzymatic method shown to be successful at each successive coupling step for the synthesis of the oligopeptide. Bovine and chum salmon trypsins were superior to Streptomyces griseus trypsin as the catalyst.

Amides↗
PubMed1995

Elevated serum levels of immunoreactive anionic trypsin (but not cationic trypsin) signals pancreatic disease.

Pancreatic juice from most studied species contains two major forms of trypsin, one with anionic electrophoretic mobility and one with cationic mobility. They are referred to as anionic and cationic trypsin(ogen). The purpose of this study was to measure immunoreactive anionic trypsin (irAT) and immunoreactive cationic trypsin (irCT) in sera from patients with pancreatic cancer (n = 39) and chronic pancreatitis (n = 32) using two specific ELISA methods. Sera from 72 healthy persons were used as controls. Patients with pancreatic cancer showed significantly elevated serum levels of irAT median level 39 vs 20.5 micrograms/L in the control group (p < 0.001). No differences in irCT levels were found. The ratio between irAT and irCT in serum was significantly increased (p < 0.001). Patients with chronic pancreatitis showed a wide range of both irAT and irCT levels, but no significant differences compared to the control group. The ratio between irAT and irCT was, however, significantly increased also in this group of patients. The results suggest a nonparallel secretion of anionic and cationic trypsinogen in pancreatic disease. This is a pattern that has been observed in experimental forms of chronic "hyperCCKemia."

Aged↗
PubMed1991

Geometry of binding of the N alpha-tosylated piperidides of m-amidino-, p-amidino- and p-guanidino phenylalanine to thrombin and trypsin. X-ray crystal structures of their trypsin complexes and modeling of their thrombin complexes.

The X-ray crystal structures of the complexes formed with bovine trypsin and the N alpha-tosylated piperidides of m-amidino-, p-amidino- and p-guanidino-D,L-phenylalanine (3-TAPAP, 4-TAPAP and 4-TGPAP) were determined with data to 1.8 A resolution. The L-stereoisomer of 3-TAPAP binds as a compact entity into the active site of trypsin, with the amidino and the carbonyl groups of the central amidinophenylalanyl residue hydrogen-bonded to Gly216 of trypsin. According to modeling and energy minimization, 3-TAPAP fits perfectly in this conformation to the more restrictive thrombin active site also (Bajusz et al. (1978) Int. J. Pept. Prot. Res. 12, 217-221); the piperidine moiety extends into the cage-like S2 subsite of thrombin, but leaves room for additional substituents which might help to improve binding and pharmacological properties. In contrast, 4-TAPAP and 4-TGPAP bind only weakly and in an extended conformation to trypsin; their considerably enhanced affinities for thrombin would suggest a more compact binding to thrombin.

Amidines↗