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PubMed2004

Molecular characterisation of five trypsin-like peptidase transcripts from the salmon louse (Lepeophtheirus salmonis) intestine.

Four novel trypsin-like S1A peptidase transcripts (LsTryp2-5) from the marine parasitic copepod Lepeophtheirus salmonis were characterised based on analyses of 1918 expressed sequence tags from two adult female libraries. In addition, one previously described salmon louse trypsin, LsTryp1, has been further characterised. The five peptidases possessed all residues typically found in trypsins in correct sequence contexts. Interestingly, two cysteine residues, possibly involved in a disulphide bridge not previously reported in trypsins are conserved in all louse trypsin sequences. Phylogenetic analyses showed that the five louse peptidases form a monophyletic group with other crustacean trypsins (Brachyurin Ts). Quantitative PCR analyses demonstrated increased transcript levels from planktonic to early host-attached stages and from preadult to sexually mature adult stages. Furthermore, sex-specific differences in transcription regulation were found. In situ hybridisation demonstrated that all five trypsin-like peptidases are transcribed throughout the undifferentiated midgut, indicating a digestive function. The sequence characteristics, histological localisation and transcript regulation suggest that LsTryp1-4 encode typical digestive trypsins. LsTryp5, however, showed some sequence and regulatory peculiarities that rendered its function less clear. Our findings support earlier suggestions for the function of the midgut cells and suggest the existence of an additional undifferentiated cell-type.

Amino Acid Sequence↗
PubMed2003

Structural consequences of accommodation of four non-cognate amino acid residues in the S1 pocket of bovine trypsin and chymotrypsin.

Crystal structures of P1 Gly, Val, Leu and Phe bovine pancreatic trypsin inhibitor (BPTI) variants in complex with two serine proteinases, bovine trypsin and chymotrypsin, have been determined. The association constants for the four mutants with the two enzymes show that the enlargement of the volume of the P1 residue is accompanied by an increase of the binding energy, which is more pronounced for bovine chymotrypsin. Since the conformation of the P1 side-chains in the two S1 pockets is very similar, we suggest that the difference in DeltaG values between the enzymes must arise from the more polar environment of the S1 site of trypsin. This results mainly from the substitutions of Met192 and Ser189 observed in chymotrypsin with Gln192 and Asp189 present in trypsin. The more polar interior of the S1 site of trypsin is reflected by a much higher order of the solvent network in the empty pocket of the enzyme, as is observed in the complexes of the two enzymes with the P1 Gly BPTI variant. The more optimal binding of the large hydrophobic P1 residues by chymotrypsin is also reflected by shrinkage of the S1 pocket upon the accommodation of the cognate residues of this enzyme. Conversely, the S1 pocket of trypsin expands upon binding of such side-chains, possibly to avoid interaction with the polar residues of the walls. Further differentiation between the two enzymes is achieved by small differences in the shape of the S1 sites, resulting in an unequal steric hindrance of some of the side-chains, as observed for the gamma-branched P1 Leu variant of BPTI, which is much more favored by bovine chymotrypsin than trypsin. Analysis of the discrimination of beta-branched residues by trypsin and chymotrypsin is based on the complexes with the P1 Val BPTI variant. Steric repulsion of the P1 Val residue by the walls of the S1 pocket of both enzymes prevents the P1 Val side-chain from adopting the most optimal chi1 value.

Amino Acids↗
PubMed2005

Evidence that protease-activated receptor-2 mediates trypsin-induced reversal of stellation in cultured rat astrocytes.

Serine proteases such as thrombin and trypsin play a key role in the development and repair processes in the central nervous system. Molecular actions of serine proteases include multiple cellular events like activation of protease-activated receptors (PARs). PARs belong to a family of G protein-coupled receptors that can be stimulated through their proteolytic cleavage by ligands. PAR-2 has been implicated in neurodegenerative diseases including astrogliosis. Although recent studies have shown that low concentration of trypsin activates PAR-2, its role in morphological changes in primary astrocytes has not been studied. In the present study, we investigated the effects of PAR-2 in astrocyte stellation in rat primary astrocyte culture. Both trypsin (0.1-1 U/ml) and a PAR-2-activating peptide SLIGRL-NH2 (1-50 microM) significantly reversed the stellation induced by serum deprivation in rat astrocytes. Treatment of astrocytes with trypsin or SLIGRL-NH2 resulted in a transient rise of the intracellular Ca2+ level and trypsin-induced morphological changes were blocked by BAPTA, a Ca2+ chelator. In addition, a protein kinase C (PKC) inhibitor, bisindolylmaleimide significantly inhibited the trypsin-induced morphological changes, whereas activation of PKC by phorbol-12-myristate-13-acetate acted as trypsin. Taken together, these results suggest that activation of PAR-2 by trypsin caused reversal of stellation in cultured astrocytes, in part, via the mobilization of intracellular Ca2+ and activation of PKC.

Animals↗
PubMed2004

Effects of ultrasound and additives on the function and structure of trypsin.

Encapsulating proteins in polymeric microspheres is a useful mode of drug delivery, but the proteins are subjected to damage in the process of ultrasound emulsion microencapsulation. The objective of this study was to investigate the effects of ultrasound power and duration on the function and structure of trypsin, and the reason of protein denaturation when it was irradiated by 20 kHz ultrasound. The relatively stable enzyme, trypsin, was dissolved in aqueous solution in the presence and absence of additives to study the stability of trypsin during the ultrasound irradiation. The damage of the molecular structure of trypsin was detected via combined high performance liquid chromatogram and electrospray ionization mass spectrometry (HPLC-ESI-MS). The results showed that the activity of trypsin decreased with increasing ultrasound power from 100 to 500 W or extending the irradiation time from 1 to 20 min. This effect could be enhanced via aerating the solution for a duration 10 min at 300 W. Fragments of trypsin were detected in the treatment (300 W, 10 min) by HPLC-ESI-MS. The additives, Tween 80 and mannitol, could protect trypsin against the inactivation caused by ultrasound. The reason of inactivation was partly from the alteration of the molecular conformation and partly from the modification or damage of trypsin's molecular structure.

Drug Compounding↗
PubMed1998

Expression of trypsin by epithelial cells of various tissues, leukocytes, and neurons in human and mouse.

It has long been believed that trypsin is normally synthesized only in the pancreas. In the present study, expression of trypsin in human and mouse nonpancreatic tissues was examined. Northern blot analysis of normal human tissues indicated that the trypsin gene is expressed at high levels in the pancreas and spleen and considerably in the small intestine. However, in situ hybridization and immunohistochemistry demonstrated that trypsin is widely expressed in epithelial cells of the skin, esophagus, stomach, small intestine, lung, kidney, liver, and extrahepatic bile duct, as well as splenic and neuronal cells. In the spleen, trypsin message was detected in macrophages, monocytes, and lymphocytes in the white pulp. In the brain, it was detected in the nerve cells of the hippocampus and cerebral cortex. Analysis by gelatin zymography confirmed the presence of a latent or an active form of trypsin in various normal mouse tissues. Reverse transcription-polymerase chain reaction analysis also confirmed the expression of trypsin genes in the spleen, liver, kidney, and brain of normal mice. Such a broad distribution of trypsin suggests its general roles in the maintenance of normal epithelial cell functions, the immune defense system, and the central nervous system.

Animals↗
PubMed1997

Purification and partial characterization of feline trypsin.

Trypsins have been purified and characterized in a multitude of mammalian and nonmammalian species, but not in the domestic cat (Felis catus). In several species two or more isoforms of trypsin have been isolated. Feline trypsin was purified from feline pancrease by sulfuric acid extraction, ammonium sulfate fractionation, gel filtration and affinity chromatography on a benzamidine-activated sepharose gel. Feline trypsinogen was purified by sulfuric acid extraction, SDS-PAGE and electroelution. Only one isoform of feline trypsin and its zymogen could be demonstrated. Isoelectric focusing on agarose gel revealed an isoelectric point of greater than 10.0 for both feline trypsinogen and trypsin. The molecular weight of feline trysinogen was estimated at 22,600, while the molecular weight of feline trypsin was estimated at 21,000. The N-terminal amino acid sequence of feline trypsinogen was Phe-Pro-Ile-Asp-Asp-Asp-Asp-Lys-Ile-Val-Gly-Gly-Tyr-Thr-Asn-Arg. We conclude that cats either have only one isoform of trypsin or that other isoforms are present in minute quantities, undetectable by commonly used methods. We further conclude that feline trypsinogen and trypsin are cationic. Finally, the N-terminal amino acid sequence of the last 16 amino acid residues of feline trypsinogen is closely related to that of other mammalian species and the final 8 amino acid residues (termed trypsinogen activation peptide) are identical to those of canine cationic trypsinogen.

Amino Acid Sequence↗
PubMed1999

Evaluation of the effects of different trypsin treatments on semen quality after BHV-1 inactivation, and a comparison of the results before and after freezing, assessed by a computer image analyzer.

Semen infected experimentally with infectious bovine rhinotracheitis virus (BHV-1) was treated with trypsin at concentrations of 0.30%, 0.25% and 0.15%, with or without (w or w/o) trypsin inhibitor in order to render the semen virus free. The trypsin treatments (at 0.30% and 0.25% by concentration) inactivating the virus up to 10(4) TCID50/ml, and its effects on semen quality were assessed weekly from the 1st to 20th week after being frozen. The following parameters were determined using a computerized semen analysis system (Hamilton Thorn motility analyzer, HTM): total motility, progressive motility and linearity of sperm cells. The results showed that the total and progressive motility of sperm cells were reduced in frozen/thawed semen, principally in the semen treated with trypsin at concentrations of 0.30%. Moreover, the plasma membranes were damaged by trypsin treatments (0.30% by concentration), as determined by the hypoosmotic swelling test (HOS test). These findings suggest that trypsin treatments were effective against the virus however the effects on semen quality and the possibility of a decrease in semen fertility were clear. Trypsin treatment could be recommended at a maximum concentration of 0.25% (w/o trypsin inhibitor) on semen with a high concentration and high motility values of spermatozoa before freezing.

Animals↗
PubMed2003

Molecular cloning of trypsin-like cDNAs and comparison of proteinase activities in the salivary glands and gut of the tarnished plant bug Lygus lineolaris (Heteroptera: Miridae).

Using specific proteinase inhibitors, we demonstrated that serine proteinases in the tarnished plant bug, Lygus lineolaris, are major proteinases in both salivary glands and gut tissues. Gut proteinases were less sensitive to inhibition than proteinases from the salivary glands. Up to 80% azocaseinase and 90% of BApNAse activities in the salivary glands were inhibited by aprotinin, benzamidine, and PMSF, whereas only 46% azocaseinase and 60% BApNAse activities in the gut were suppressed by benzamidine, leupeptin, and TLCK. The pH optima for azocaseinase activity in salivary glands ranged from 6.2 to 10.6, whereas the pH optima for gut proteinases was acidic for general and alkaline for tryptic proteinases. Zymogram analysis demonstrated that approximately 26-kDa proteinases from salivary glands were active against both gelatin and casein substrates. Three trypsin-like cDNAs, LlSgP2-4, and one trypsin-like cDNA, L1GtP1, were cloned from salivary glands and gut, respectively. Putative trypsin precursors from all cloned cDNAs contained a signal peptide, activation peptide, and conserved N-termini (IVGG). Other structural features included His, Asp, and Ser residues for the catalytic amino acid triad of serine proteinase active sites, residues for the binding pocket, and four pairs of cysteine residues for disulfide bridges. Deduced trypsin-like proteins from LlSgP2, LlSgP3, and LlGtP1 cDNAs shared 98-99% sequence identity with a previously reported trypsin-like precursor, whereas the trypsin-like protein of LlSgP4 shared only 44% sequence identity with all other trypsin-like proteins, indicating multi-trypsin forms are present in L. lineolaris.

Amino Acid Sequence↗
PubMed1992

Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214.

Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Asp102. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser214 adjacent to Asp102. Ser214 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-A crystal structure (R = 0.150) of trypsin S214K shows that Lys214 occupies the position held by Ser214 and a buried water molecule in the buried polar cave. Lys214-N zeta is solvent inaccessible and is less than 5 A from the catalytic Asp102. The side chain of Glu214 (2.8 A, R = 0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Asp102. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter kcat demonstrates that, compared to trypsin, S214K has 1% of the catalytic activity on a tripeptide amide substrate and S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Asp102.

Amino Acid Sequence↗
PubMed1994

Exogenous acetate reconstitutes the enzymatic activity of trypsin Asp189Ser.

The specificity of trypsin for Arg- and Lys-containing substrates depends upon the presence of Asp189 at the base of the primary binding pocket. The crystal structure of anionic rat trypsin D189S complexed with BPTI reveals that removal of the aspartate side chain permits the binding of a well-ordered acetate ion in a similar position. The acetate makes polar interactions with Gly226, Tyr228, and several water molecules and is further accommodated by rotation of the Ser189 side chain out of the binding pocket. The carboxylate group of the acetate anion is oriented toward the substrate in a manner similar to that of Asp189 and Asp226 in wild-type trypsin and trypsin D189G/G226D. Evaluation of kinetic parameters for amide substrate cleavage by trypsin D189S shows that high concentrations of acetate increase the catalytic efficiency of the enzyme by as much as 300-fold. Under these conditions, the rate of substrate turnover toward a peptidylarginine amide substrate equals that of wild-type trypsin. These data demonstrate that the well-established requirement for a negatively charged moiety at the base of the trypsin specificity pocket may be fulfilled by a noncovalently bound ligand. The binding pocket of this variant maintains a trypsin-like conformation, explaining the inability of the mutant enzyme to efficiently hydrolyze chymotrypsin substrates possessing Phe in the P1 position.

Acetates↗
PubMed1993

Structural characterization of the trypsinized estrogen receptor.

Structural differences between the unoccupied and ligand-occupied rat uterine estrogen receptors (ERs) were investigated using partial proteolysis followed by immunoblotting, affinity labeling, and gel filtration chromatography. Trypsin digestion of the unoccupied ER at 4 degrees C resulted in retention of 70-80% of high-affinity [3H]estradiol binding. Only two fragments of the rat ER were detected after prolonged trypsin treatment of the unoccupied ER followed by affinity labeling with [3H]tamoxifen aziridine. One fragment represents the intact steroid binding domain (28 kDa), and the other fragment is about 10 kDa. The small 10-kDa fragment of the ER detected by denaturing gel electrophoresis is shown to be held in a large oligomeric complex in solution using gel filtration chromatography. This oligomeric complex probably represents the steroid binding domain, which has its tertiary structure maintained predominantly by noncovalent interactions between the trypsin-generated fragments. The estrogen, anti-estrogen, and unoccupied trypsinized ERs all result in similar patterns of fragments after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detection by immunoblotting. Although no new trypsin cleavage sites were exposed, the sensitivity of the available trypsin sites was altered by heating the ER and, to a lesser extent, by hormone treatment. Gel filtration chromatography of the trypsinized estradiol- and 4-hydroxytamoxifen-occupied ERs demonstrates similar, diffuse peaks centered at about the correct size for the intact steroid binding domain (28 kDa), whereas the trypsinized unoccupied ER results in a sharp, discrete peak centered at about 80 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Crystal structures of two engineered thiol trypsins.

We have determined the three-dimensional structures of engineered rat trypsins which mimic the active sites of two classes of cysteine proteases. The catalytic serine was replaced with cysteine (S195C) to test the ability of sulfur to function as a nucleophile in a serine protease environment. This variant mimics the cysteine trypsin class of thiol proteases. An additional mutation of the active site aspartate to an asparagine (D102N) created the catalytic triad of the papain-type cysteine proteases. Rat trypsins S195C and D102N,S195C were solved to 2.5 and 2.0 A, respectively. The refined structures were analyzed to determine the structural basis for the 10(6)-fold loss of activity of trypsin S195C and the 10(8)-fold loss of activity of trypsin D102N,S195C, relative to rat trypsin. The active site thiols were found in a reduced state in contrast to the oxidized thiols found in previous thiol protease structures. These are the first reported structures of serine proteases with the catalytic centers of sulfhydryl proteases. Structure analysis revealed only subtle global changes in enzyme conformation. The substrate binding pocket is unaltered, and active site amino acid 102 forms hydrogen bonds to H57 and S214 as well as to the backbone amides of A56 and H57. In trypsin S195C, D102 is a hydrogen-bond acceptor for H57 which allows the other imidazole nitrogen to function as a base during catalysis. In trypsin D102N,S195C, the asparagine at position 102 is a hydrogen-bond donor to H57 which places a proton on the imidazole nitrogen proximal to the nucleophile. This tautomer of H57 is unable to act as a base in catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1979

Transfer of singlet energy within trypsin.

Transfers of singlet energy within trypsin were investigated by measuring the fluorescence absorption anisotropy of its tryptophan residues. A ratio of the anisotropy of trypsin to that for N-acetyl-L-tryptophanamide was determined between 306 and 250 nm. The ratio had an average value of 0.7, whether the trypsin anisotropy was measured at 228 of 296 K. However, trypsin dissolved in 5 M guanidine hydrochloride showed little fluorescence depolarization at 228 K (the anisotropy ratio was approximately equal to 0.9). Thus, there is an extensive conformation-dependent energy transfer between tryptophans in trypsin. The ratio of anisotropies of tyrpsin at 304--270 nm was used to estimate energy transfer from tyrosine to tryptophan. Ratios of 1.8 and 1.7 were obtained at 296 K for the native and guanidinium-unfolded enzyme, respectively. The comparable value for N-acetyl-L-tryptophanamide was 1.7. This indicates that there is little transfer from tyrosine to tryptophan in trypsin at 296 K. As confirmation, the excitation wavelength dependencies of the indole fluorescence quantum yield were the same for native and unfolded trypsin. When experiments were performed at 228 K, the 304--270-nm anisotropy ratios were 2.6 for native and 2.1 for unfolded trypsin at pH2. This indicates that the efficiency of energy transfer from tyrosine to tryptophan increases at low temperatures. A photochemical source of error in the quantitation of the efficiency of energy transfer from tyrosine to tryptophan is also described.

Energy Transfer↗
PubMed1996

Delocalizing trypsin specificity with metal activation.

Recognition for proteolysis by trypsin depends almost exclusively on tight binding of arginine or lysine side chains by the primary substrate specificity pocket. Although extended subsite interactions are important for catalysis, the majority of binding energy is localized in the P1 pocket. Analysis of the interactions of trypsin with the P1 residue of the bound inhibitors ecotin and bovine pancreatic trypsin inhibitor suggested that the mutation D189S would improve metal-assisted trypsin N143H, E151H specificity toward peptides that have a Tyr at P1 and a His at P2'. In the presence of transition metals, the catalytic efficiency of the triple mutant Tn N143H, E151H, D189S improved toward the tyrosine-containing peptide AGPYAHSS. Trypsin N143H, E151H, D189S exhibits a 25-fold increase in activity with nickel and a 150-fold increase in activity with zinc relative to trypsin N143H, E151H on this peptide. In addition, activity of trypsin N143H, E151H, D189S toward an arginine-containing peptide, YLVGPRGHFYDA, is enhanced by copper, nickel, and zinc. With this substrate, copper yields a 30-fold, nickel a 70-fold, and zinc a 350-fold increase in activity over background hydrolysis without metal. These results demonstrate that the engineering of multiple substrate binding subsites in trypsin can be used to delocalize protease specificity by increasing relative substrate binding contributions from alternate engineered subsites.

Amino Acid Sequence↗
PubMed2004

cDNA clone of a putative peanut (Arachis hypogaea L.) trypsin inhibitor has homology with peanut allergens Ara h 3 and Ara h 4.

Trypsin inhibitors are pathogenesis-related (PR) proteins, which play an important role in the plant defense mechanism against insects and pathogens. Peanut trypsin inhibitors are low molecular mass seed storage proteins. Like peanut allergens, they are stable to acid and heat, resistant to digestion, and can have a negative impact on human health. In peanut, five Bowman-Birk trypsin inhibitors (BBTI) have been isolated and amino acid sequences published. However, to date, no peanut BBTI sequence is available at both the cDNA and the genomic levels. The objectives of this investigation were (i) to synthesize degenerate oligonucleotides based on conserved regions of published amino acid sequences of BBTI, BII, and BIII; (ii) to isolate, sequence, and analyze at least one positive peanut trypsin inhibitor cDNA clone using the synthesized (32)P-labeled oligonucleotides as probes; and (iii) to determine its trypsin inhibitory activity. Thirty-two degenerate oligonucleotides DNA primers of 24 nucleotides each were synthesized based on the published amino acid sequences of peanut BBTI, and two were selected as probes to screen a peanut Lambda gt 11 cDNA library. Three putative positive clones were isolated, purified, and subcloned, and one was sequenced. Sequence analysis revealed a partial cDNA clone of 643 bp with a start codon. This clone shares 93 and 96% nucleotide sequence homology with peanut allergens Ara h 3 and Ara h 4 cDNA clones, respectively. A trypsin inhibitor assay revealed that peanut allergen Ara h 3 has a trypsin inhibitory activity of 11 238 TIA/mg protein. We concluded that peanut allergen Ara h 3 may also function as a trypsin inhibitor.

Allergens↗
PubMed1999

Nutritive value and effect of blanching on the trypsin and chymotrypsin inhibitor activities of selected leafy vegetables.

Proximate composition, energy, mineral and vitamin contents and the effect of blanching methods and times on the trypsin and chymotrypsin inhibitor activities were studied using cabbage, collard, turnip, peanut, and sweet potato leaves. Results of this study indicated that, crude protein, crude fat, carbohydrate and ash contents were in the range of 15.5-25.6%, 1.4-6.5%, 60.4-73.1% and 6.8-7.5%, respectively. Total dietary fiber was lowest in cabbage (28.2 g/100 g) and highest in the collard leaves (43.1%) while energy content per 100 g of vegetables was highest in sweet potato leaves (402 kcal) and lowest in cabbage (379 kcal). The mineral content per 100 g of vegetables were in the range of 33.4-249.8 mg, 241.2-471.2 mg, 12.1-75.1 mg, 14.9-98.9 mg, 0.5-3.5 mg and 0.9-3.1 mg for Ca, K, Na, Mg, Fe and Zn, respectively. For ascorbic acid, riboflavin, thiamin and total carotenoids, concentrations in 100 g of vegetables were in the range of 45.1-112.7 mg, 0.2-0.3 mg, 0.3-0.8 mg and 2.0-7.3 mg, respectively. The trypsin inhibitory activity per gram of the vegetables was highest in collard (60.1 TIU/g) and lowest in peanut leaves (41.0 TIU/g). Chymotrypsin inhibitor activity was highest in the peanut (69.6 CIU/g) but lowest in the collard leaves (48.0 CIU/g). Both trypsin and chymotrypsin inhibitor activities were significantly (p < 0.05) reduced by most of the treatments in either the conventional or microwave blanching methods. In the conventional blanching method, trypsin inhibitor activity was reduced by 0.5, 6.8, 11.9, 9.0 and 19.3 percent in cabbage, collard, turnip, sweet potato and peanut leaves, respectively, when the vegetables were blanched for 2.5 minutes but after blanching for 10 minutes, the trypsin inhibitor activity was reduced by 29.7, 34.9, 54.3, 52.3 and 65.6 percent in cabbage, collard, turnip, sweet potato and peanut greens, respectively. For the microwave oven blanching, trypsin inhibitor activity was reduced by 3.8, 3.3, 32.7, 5.0 and 9.5 percent in cabbage, collard, turnip, sweet potato and peanut leaves, respectively when the vegetables were blanched for 30 seconds. When blanched for 60 seconds, trypsin inhibitor activity was reduced by 16.2, 45.8, 46.2, 51.0 and 42.4 percent in cabbage, collard, turnip, sweet potato and peanut greens, respectively. Similar trends in the reduction of chymotrypsin inhibitor activity were observed when the vegetables were conventionally blanched for 2.5, 5 and 10 minutes and when blanched by microwave oven for 30, 45 and 60 seconds. Based on the results of this study, the vegetables were good dietary sources of minerals, vitamins, carbohydrate and proteins. Also, blanching was an effective method for reducing the trypsin and chymotripsin inhibitor activities in the leafy vegetables, however, further investigation on the heating times for both conventional and microwave blanching methods is suggested.

Chymotrypsin↗
PubMed1998

The complete amino acid sequence of a trypsin inhibitor from Bauhinia variegata var. candida seeds.

Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with Mr of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show Ki values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)-Ile (64).

Amino Acid Sequence↗
PubMed1982

Mechanism of inactivation of trypsin by antithrombin.

General aspects of the mechanism of antithrombin action were elucidated by a comparison of the inactivation of trypsin by antithrombin with the inactivation of coagulation proteinases by the inhibitor. Bovine antithrombin and bovine trypsin were shown to form an inactive equimolar complex. A non-complexed, proteolytically modified form of antithrombin, electrophoretically identical with that formed in the reaction with coagulation proteinases, was also produced in the reaction with trypsin. In the absence of heparin, the inactivation of trypsin by antithrombin was 20 times faster than the inactivation of thrombin; the second-order rate constant was 1.5 x 10(5)m(-1).s(-1) at 25 degrees C and pH 7.4. However, the inhibition of thrombin was accelerated about 30 times more efficiently by small amounts of heparin than was trypsin inhibition. Dissociation of the antithrombin-trypsin complex at pH 7.4 followed first-order kinetics with a half-life for the complex of about 80h at 25 degrees C. The complex was rapidly and quantitatively dissociated at pH 11, resulting in the liberation of a modified two-chain form of the inhibitor, cleaved at the same Arg-Ser bond as in modified antithrombin released from complexes with thrombin, Factor Xa and Factor IXa. This supports the previous proposal that this bond is the active-site bond of antithrombin. Antisera specific for thrombin-modified antithrombin reacted with purified antithrombin-trypsin complex, indicating that the inhibitor was present in the complex in a form immunologically identical with thrombin-modified antithrombin. The results thus suggest a common mechanism, but different kinetics, for the inhibition of trypsin and coagulation proteinases by antithrombin.

Amino Acid Sequence↗