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PubMed1993

Trypsin immobilization on to polymer surface through grafted layer and its reaction with inhibitors.

Trypsin was covalently immobilized and physically adsorbed on to the surface of poly(ethylene terephthalate) fibres using poly(acrylic acid) (PAAc) chains grafted on to the ozonized fibres. The covalent immobilization was accomplished through amide formation between amino groups of trypsin and carboxyl groups of grafted PAAc chains, with the use of water-soluble carbodiimide. A set of samples with surface concentrations of grafted polymer ranging from 0.03 to 2.5 micrograms/cm2 was used to study the effects of grafted layer on the enzymatic activity of immobilized trypsin and its inhibition by trypsin inhibitors of different molecular sizes. The amount of immobilized trypsin increased linearly with an increase in graft yield of fibres, but the activity of immobilized enzyme reached saturation at a certain graft yield, probably because of diffusion limitation for the transport of enzyme substrate molecules into the grafted PAAc layer. The reduction of inhibition with an increase in graft yield and in molecular weight of inhibitors was attributed to enhancement of steric hindrance and enzyme inactivation in the dense grafted layer. We also found that the adsorbed trypsin was inhibited more easily than the covalently immobilized at any concentration of the grafted PAAc and for any type of inhibitor used.

Acrylic Resins↗
PubMed1982

The effect of alpha 2-macroglobulin on the interaction of alpha 1-proteinase inhibitor with porcine trypsin.

The rate of dissociation of the alpha 1-proteinase inhibitor:porcine trypsin complex was compared with that in the presence of alpha 2-macroglobulin. In the presence of the latter inhibitor the dissociation was more rapid and active alpha 1-proteinase inhibitor could be recovered in the mixture. However, no active inhibitor could be detected after dissociation in the absence of alpha 2-macroglobulin. This recovery of active alpha 1-proteinase inhibitor from complexes with porcine trypsin is the first demonstration of a thermodynamic equilibrium between this inhibitor and proteinase. Consequently, the transfer of trypsin from complexes with alpha 1-proteinase inhibitor to alpha 2-macroglobulin may be explained as a passive phenomenon which does not require a physical collision between alpha 2-macroglobulin and the alpha 1-proteinase inhibitor:porcine trypsin complex. The dissociation of the complex occurs more rapidly in the presence of alpha 2-macroglobulin because this inhibitor complexes trypsin leaving the alpha 1-proteinase inhibitor:porcine trypsin complex by both the irreversible breakdown step and by reversible dissociation of the complex.

Animals↗
PubMed1985

Purification and some properties of two anionic trypsins from the eel (Anguilla japonica).

Two anionic enzymes, designated as trypsins 1 and 2, were purified from the pancreas of the eel Anguilla japonica by DEAE-cellulose column chromatography and Sephadex G-75 gel filtration. The final preparation of trypsin 1 was homogeneous but that of trypsin 2 still contained impurities. Both enzymes had similar pH optima of near 8.3 for the hydrolysis of N-tosyl-L-arginine methyl ester. Trypsin 1 was stabilized by calcium ions but the stability of trypsin 2 was not affected by calcium ions. Both enzymes were inhibited by typical trypsin inhibitors including serine proteinase inhibitors.

Anguilla↗
PubMed1989

Detection of trypsin- and chymotrypsin-like proteases using p-nitroanilide substrates after sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Specific chromogenic p-nitroanilide substrates have proved useful for localizing proteolytic enzymes, such as trypsin, chymotrypsin and elastase after separation by agarose gel electrophoresis and when immobilized on nitrocellulose. This procedure was further developed for use with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). After SDS-PAGE, proteins were transferred electrophoretically to a nitrocellulose membrane. The membrane was incubated for 10-60 min with Bz-Ile-Glu-Gly-Arg-p-nitroanilide as a substrate for detection of trypsin-like proteases and with MeO-Suc-Arg-Pro-Tyr-p-nitroanilide for detection of chymotrypsin. The yellow p-nitroanilide released at the site of proteolytic activity was converted into a visible and stable red azo dye. By this method was identified and determined the molecular weight of a trypsin-like protease that occurs at high concentrations in mucinous ovarian tumour cyst fluid together with its specific inhibitor peptide, tumour-associated trypsin inhibitor (TATI). The method was also used to visualize trypsin and chymotrypsin in human pancreatic juice. Using the trypsin substrate, three proteolytic bands, corresponding to Mr of 22,000, 24,000 and 26,000 daltons, were visualized in pancreatic juice, while the proteolytic zones in cyst fluid had Mr of 25,000 and 28,000 daltons. With the chymotrypsin substrate, a band of 29,000 daltons was visualized in pancreatic juice, whereas no activity was detected in cyst fluid. By incubation of the blotted cyst fluid proteins with 125I-labelled TATI, a pattern of bands at 25,000 and 28,000 daltons was detected identical to that obtained with the chromogenic substrate.

Anilides↗
PubMed1983

Model study of hydrophobic interactions of alpha- and beta-trypsin and alpha-chymotrypsin.

The hydrophobic interactions of alpha- and beta-trypsin as a function of ionic strength and pH were studied by hydrophobic chromatography. Evidence was obtained that in spite of the identical specificities and similar activities of alpha- and beta-trypsin, the cleavage of the Lys-Ser bond induces conformational changes in the neighbourhood of the active site. Over a wide range of pH and salt concentration the non-polar residues on the surface of the molecule of beta-trypsin are more exposed to an external environment than on the molecule of alpha-trypsin. In the trypsin(chymotrypsin)-inhibitor complexes the majority of hydrophobic amino acids are buried; other hydrophobic residues localized on the surface contribute only very slightly to the interaction with the chromatographic support. The retention of trypsin, chymotrypsin and their diisopropylphosphoryl derivatives on a support with flexible hydrophobic ligands bonded to the matrix through a spacer (octyl-Sepharose) was correlated with the retention on a support with hydrophobic binding sites incorporated into the rigid matrix of the resin (Spheron). The native enzymes are always more retained; this indicates that the substitution results in the shielding of the non-polar residues in the neighbourhood of the active site. The differences in the slope of individual proteins, resulting from the correlation of the retention values obtained with both supports at several sodium chloride concentrations are explained by differences in the accessibility of the surface non-polar residues in the individual proteins. In experiments with model peptides the contribution of the individual hydrophobic amino acids to the retention was investigated.

Animals↗
PubMed1996

Hydrophobic interactions control zymogen activation in the trypsin family of serine proteases.

Trypsinogen is converted to trypsin by the removal of a peptide from the N terminus, which permits formation of a salt bridge between the new N-terminal Ile (residue 16) and Asp194. Formation of this salt bridge triggers a conformational change in the "activation domain" of trypsin, creating the S1 binding site and oxyanion hole. Thus, the activation of trypsinogen appears to represent an example of protein folding driven by electrostatic interactions. The following trypsin mutants have been constructed to explore this problem: Asp194Asn, Ile16Val, Ile16Ala, and Ile16Gly. The bovine pancreatic trypsin inhibitor (BPTI), benzamidine, and leupeptin affinities and activity and pH-rate profiles of these mutants have been measured. The changes in BPTI and benzamidine affinity measure destabilization of the activation domain. These experiments indicate that hydrophobic interactions of the Ile16 side chain provide 5 kcal/mol of stabilization energy to the activation domain while the salt bridge accounts for 3 kcal/mol. Thus, hydrophobic interactions provide the majority of stabilization energy for the trypsinogen to trypsin conversion. The pH-rate profiles of I16A and I16G are significantly different than the pH-rate profile of trypsin, further confirming that the activation domain has been destabilized. Moreover, these mutations decrease kcat/Km and leupeptin affinity in parallel with the decrease in stability of the activation domain. Acylation is selectively decreased, while substrate binding and deacylation are not affected. Together these observations indicate that the stability of protein structure is an important component of transition state stabilization in enzyme catalysis. These results also suggest that active zymogens can be created without providing a counterion for Asp194, and thus have important implications for the elucidation of the structural features which account for the zymogen activity of tissue plasminogen activator and urokinase.

Amino Acid Sequence↗
PubMed2006

Purification and characterization of trypsin from the spleen of tongol tuna (Thunnus tonggol).

Trypsin from tongol tuna (Thunnus tonggol) spleen was purified to 402-fold by ammonium sulfate precipitation, followed by a series of chromatographic separations. The molecular mass of trypsin was estimated to be 24 kDa by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin appearing as a single band on native PAGE showed the maximal activity at pH 8.5 and 65 degrees C. It was stable in a wide pH range of 6-11 but unstable at the temperatures greater than 50 degrees C. The enzyme required calcium ion for thermal stability. The activity was strongly inhibited by 1.0 g/L soybean trypsin inhibitor and 5 mM TLCK and partially inhibited by 2 mM ethylenediaminetetraacetic acid. Activity was lowered with an increasing NaCl concentration (0-30%). The enzyme had a Km for Nalpha-p-tosyl-L-arginine methyl ester hydrochloride of 0.25 mM and a Kcat of 200 s-1. The N-terminal amino acid sequence of trypsin was determined as IVGGYECQAHSQPHQVSLNA and was very homologous to other trypsins.

Amino Acid Sequence↗
PubMed1998

Structural and functional analyses of benzamidine-based inhibitors in complex with trypsin: implications for the inhibition of factor Xa, tPA, and urokinase.

The trypsin-like serine proteinase superfamily contains a number of potential therapeutic targets, many of which are unsuitable for routine X-ray crystallographic studies. We have cocrystallized a selection of benzamidine-based inhibitors with bovine trypsin and solved their structures to a resolution of up to 1.7 A. Despite similar chemical formulas, the inhibitors exhibit a range of diverse binding modes that reflect their inhibitory spectra against the serine proteinases trypsin, thrombin, factor Xa, tissue-type plasminogen activator (tPA) and urokinase (uPA). In contrast to the compact folded conformations of thrombin inhibitors which allow optimal binding in the well-defined hydrophobic S2/S4 pocket of thrombin, those effective against factor Xa exhibit an extended conformation that allows occupation of the S3/S4 region, where hydrophobic and electrostatic interactions can stabilize the conformation. One group of inhibitors containing an N-terminal 2,4, 6-triisopropylphenylsulfonyl (TIPPS) moiety show little or no penetration into the S3/S4 subsites of trypsin. These latter sites are occluded in uPA, explaining why this class of compounds is effective against uPA. Despite presenting an extensive hydrophobic surface toward the solvent, the Ki values for TIPPS-containing compounds against trypsin is in the range 10(-7) to 10(-8) M. Comparison of the binding of a bis-benzamidine inhibitor in trypsin and tPA indicate that a shift in potency can be induced by relatively minor changes in binding mode. Implications for the inhibition of these proteinases are discussed.

Animals↗
PubMed2001

Kinetic and structural properties of two isoforms of trypsin isolated from the viscera of Japanese anchovy, Engraulis japonicus.

Two isoforms of anchovy trypsin (aT-I and aT-II) were purified from the visceral extracts by (NH4)2SO4 fractionation followed by affinity chromatography, gel filtration, and ion-exchange chromatography. The homogeneity of the purified preparation was evidenced by both native- and SDS-PAGE, and further by gelatin zymography. Identities of aT-I and aT-II as trypsins were established by N-terminal amino acid sequencing, which matched exactly to the corresponding stretches of their respective amino acid sequences obtained by molecular cloning [Ahsan et al. (2000), Marine Biotechnol., in press]. Both isoforms were completely inhibited by serine protease inhibitors as well as by specific trypsin inhibitors. The purified anchovy trypsins showed considerably higher catalytic efficiencies (kcat/Km) than bovine trypsin as measured toward benzoyl-arginine p-nitroanilide (BAPA) and benzoyl-arginine ethyl ester (BAEE) at 25 degrees C; in particular, aT-II was 35 times more efficient than its mammalian counterpart against BAPA. This was due mainly to a dramatic decrease of Km values for anchovy trypsins, which are indicative of an evolutionary response toward increased substrate binding at suboptimal temperatures in the marine environment.

Amino Acid Sequence↗
PubMed1998

Cleavage of Escherichia coli tryptophan indole-lyase by trypsin at Lys406 affects the transmission of conformational changes associated with monovalent cation activation.

Escherichia coli tryptophan indole-lyase (Trpase) is a pyridoxal 5'-phosphate(pyridoxal-P)-dependent enzyme which catalyzes the hydrolytic cleavage of L-tryptophan to indole and ammonium pyruvate. This enzyme is strongly activated by K+ and similar monovalent cations, and the spectrum of the pyridoxal-P cofactor is also affected by pH and cations. Treatment of Trpase with trypsin results in a 20-100-fold decrease in elimination activity, depending on the substrate, concomitant with a change in the relative amounts of the 337 nm and 420 nm forms of the bound pyridoxal-P, and a shift in the lambda(max) from 420 nm to 423 nm. In addition, the pH sensitivity of the pyridoxal-P cofactor is eliminated after trypsin treatment. Nicked Trpase exhibits only fourfold activation by K+, compared with about 50-fold for native enzyme, but the K(A) for K+ is unaffected. Both the native and trypsin-nicked Trpase react with amino acids to form equilibrating mixtures of external aldimine and quinonoid intermediates in rapid-scanning stopped-flow experiments. However, the rate constant for quinonoid intermediate formation from L-tryptophan is reduced by at least 400-fold by treatment with trypsin. In contrast, the rate constant for formation of quinonoid intermediates of L-alanine and S-ethyl-L-cysteine is affected only twofold or less by trypsin treatment. The site of trypsin cleavage was identified by electrospray-ionization mass spectrometry as Lys406, which is predicted to lie on a flexible surface loop. Some active-site residues, particularly Arg419, which is predicted by sequence similarity to be the substrate alpha-carboxylate-binding site, and His463, are located in the sequence between Lys406 and the C-terminus. Hence, cleavage of the peptide bond of E. coli Trpase at Lys406 probably affects the change from active to inactive conformations that normally takes place in the presence of activating monovalent cations.

Binding Sites↗
PubMed1999

Primary structure and possible functions of a trypsin inhibitor of Bombyx mori.

A protein with a low molecular mass of 6027 was purified from cocoon shell of silkworm, Bombyx mori. Two-dimensional polyacrylamide gel electrophoresis (2D/PAGE) resolved this protein into a single spot with pI 4.3 and Mr 6000. Amino acid sequence analysis revealed that this protein consists of 55 amino acids, six of these being cysteine residues and is highly homologous to bovine pancreatic trypsin inhibitor-type inhibitors. The 6-kDa protein is heat stable and acid stable and inhibits bovine trypsin by forming a low-dissociation complex with trypsin in a 1 : 1 molar ratio (Ki = 2.8 x 10-10), but does not alpha-chymotrypsin. This cocoon shell-associated trypsin inhibitor (CSTI) was thus concluded to belong to the bovine pancreatic trypsin inhibitor class. CSTI was developmentally regulated in the silk gland at the final stage of larval growth, and its specific distribution in the middle silk gland, an organ in which silk proteins are stored during the final larval instar, occurred before the onset of spinning. This inhibitor protects the tryptic degradation of fibroin light (L) chain in vitro. These results suggest that this trypsin inhibitor may play an important part on regulating proteolytic activity in the silk gland or protecting silk proteins from degradation during histolysis.

Amino Acid Sequence↗
PubMed1997

Identification and expression of the cDNA-encoding human mesotrypsin(ogen), an isoform of trypsin with inhibitor resistance.

The cDNA encoding a novel isoform of human trypsinogen was identified. The isoelectric points of the proenzyme and active forms calculated from the deduced amino acid sequence are consistent with those of mesotrypsin(ogen), known to be an inhibitor-resistant trypsin isoform. The cDNA attached with a bacterial signal peptide sequence was expressed in Escherichia coli. The recombinant proenzyme purified from periplasm showed enterokinase-dependent activation similar to a major isoform of human trypsinogen. The enzyme was far less inhibited by trypsin inhibitors such as soybean trypsin inhibitor, aprotinin, or pancreatic secretory trypsin inhibitor than the control trypsin. A gel filtration assay showed that the enzyme and aprotinin did not form a stable complex. It is noteworthy that the amino acid at position 198, which is in close vicinity to the active Ser, is Arg while those of other major trypsins are all Gly. It is concluded that the cloned cDNA encodes human mesotrypsinogen, a unique isoform of trypsinogen with inhibitor resistance.

Amino Acid Sequence↗
PubMed1984

Response of rat pancreatic proteases to dietary proteins, their hydrolysates and soybean trypsin inhibitor.

The present study was designed to determine whether the adaptation of pancreatic trypsin and chymotrypsin activities to dietary protein level can be attributed to the presence in the diet of the specific peptide bonds that are substrates for these enzymes. In addition, the effect of feeding soybean trypsin inhibitor (SBTI) was tested since this compound is known to stimulate synthesis of pancreatic proteases. For 3 weeks, rats were fed a diet containing 10, 20 or 30% protein as lactalbumin (control); lactalbumin predigested by trypsin, chymotrypsin or pancreatin; a mixture of amino acids; or lactalbumin supplemented with SBTI. At the end of the experiment, the activities of trypsin, chymotrypsin and amylase were determined in the rats' pancreatic homogenates. Trypsin and chymotrypsin activities at each protein level were exactly the same whether predigested lactalbumin or lactalbumin was fed. The 20 and 30% amino acid mixtures, however, produced a decrease in activities of both proteases. SBTI doubled the activities of both proteases at all three levels of protein. Amylase activity was maximal at 20% level of protein. It is concluded that adaptation of pancreatic proteases to dietary protein level is induced by small peptides that are digestion products of dietary protein. Induction of pancreatic trypsin or chymotrypsin therefore does not require the presence of substrate for these enzymes in the intestine. Dietary SBTI produced a considerable increase in pancreatic proteases compared to that produced by proteins or peptides. It is suggested that the increases in pancreatic proteases caused either by protein-rich diets or SBTI are mediated by two distinct mechanisms.

Amylases↗
PubMed1988

Feedback regulation of stimulated pancreatic enzyme secretion during intraduodenal perfusion of trypsin in man.

The pancreatic enzyme secretion in several species is controlled by a negative feedback mechanism induced by the presence of active proteases in the duodenum. The existence of this mechanism in man is controversial. The purpose of the present study was to evaluate the effect of tryptic activity in the duodenum on phenylalanine-stimulated pancreatic enzyme secretion in healthy volunteers. A double-balloon, multi-lumen tube was used for the collection of duodenal juice containing pancreatic enzymes. The continuous infusion of phenylalanine (100 mM) into the duodenum evoked an almost constant secretion of trypsin, amylase and lipase for 160 min. The infusion of trypsin (150 mg h-1; 1.25 g l-1) caused a reduction of phenylalanine-stimulated amylase and lipase output by 25%. The subsequent infusion of aprotinin at a dose of 1.5 X 10(6) KIU for 30 min led to an almost complete inhibition of trypsin. Simultaneously, the amylase and lipase output returned to the values seen before trypsin perfusion. Infusion of a higher dose of trypsin (300 mg h-1; 2.5 g l-1) caused a more pronounced decrease in phenylalanine-stimulated amylase and lipase output by 45%. These data indicate that active trypsin in the duodenum is responsible for the inhibition of phenylalanine-stimulated pancreatic enzyme secretion in man in a dose-dependent fashion, thus confirming the existence of a feedback control of pancreatic secretion regulated by the amount of proteases in the gut.

Adolescent↗
PubMed1985

Preparation of photoreactive derivatives of trypsin-chymotrypsin inhibitors from soybeans and chick peas by selective modification of lysine residues.

Photoreactive derivatives of the Bowman-Birk trypsin-chymotrypsin inhibitor (BBI) from soybeans and of CI, the trypsin-chymotrypsin inhibitor from chick peas, were prepared by selective modification of the epsilon-amino groups of lysine residues with 2-nitro-4(5)-azidophenylsulfenyl chlorides (2,4(5)-NAPS-C1). The ultraviolet absorption spectra of the photolabeled inhibitors indicated that three out of the five lysines of BBI and one of the seven lysines of CI were modified. The inhibitory activity of the modified inhibitors towards trypsin and chymotrypsin was not reduced even after photolysis. The specific lysine residues that constitute the trypsin-inhibitory sites of BBI and CI did not react with the photoreactive reagents. Further modification of the photoreactive derivatives of BBI and CI with maleic anhydride, directed towards the trypsin-reactive sites, resulted in almost complete loss of the trypsin-inhibiting activity without reducing the ability to inhibit chymotrypsin. A pronounced potentiation effect (approximately 2x) of the chymotrypsin inhibiting activity was noted for 2,5-NAPS-CI and it was retained even after maleylation followed by photolysis, raising the possibility of exposure of an additional chymotrypsin inhibitory site in CI.

Affinity Labels↗
PubMed1971

Trypsin action on the growth of Sendai virus in tissue culture cells. I. Restoration of the infectivity for L cells by direct action of tyrpsin on L cell-borne Sendai virus.

Sendai virus grown in fertile eggs (egg Sendai) infects L cells in which the synthesis of L Sendai (grown in L cells) occurs by the one-step mechanism. L Sendai is not infectious for L cells when tested by the tube titration method although it is infectious for chick embryos. When L cells infected with egg Sendai were dispersed by trypsin and plated on a monolayer culture of L cells, the viral agents spread to the adjacent recipient cells in which the synthesis of L Sendai occurred. The newly infected L cells became infectious for L cells again by trypsin treatment. Kinetic experiments suggested that the target of trypsin is the mature virus, of L Sendai nature, just budding from the L-cell surface. By using an immunofluorescent cell-counting technique, recovery of the infectivity of L Sendai for L cells due to a direct enzymatic action of trypsin was demonstrated. Under the optimal condition, the infectivity increased 1,000-fold for L cells and 10-fold for chick embryos, and both the titers could favorably be compared. No increasing effect of trypsin was observed on the infectivity of egg Sendai. Density centrifugation studies revealed a difference between egg Sendai and L Sendai in the density. Trypsin treatment which induced the maximal enhancement of L Sendai infectivity did not affect both the densities, showing that variations of Sendai virus in the infectivity for L cells and in the density are independent types of host-controlled modification.

Animals↗
PubMed1994

In vivo binding of human inter-alpha-trypsin inhibitor free heavy chains to hyaluronic acid.

In vivo binding of human inter-alpha-trypsin inhibitor to hyaluronate was investigated by immunoelectrophoretic techniques. Pathological synovial fluids and follicular fluids both contain high concentrations of soluble hyaluronate. Heavy chain epitopes of inter-alpha-trypsin inhibitor were firmly associated with the hyaluronate in synovial fluid and follicular fluid. The hyaluronate-bound inter-alpha-trypsin inhibitor epitopes did not cross-react immunologically with bikunin. Several hyaluronate-bound inter-alpha-trypsin inhibitor fragments with molecular masses in the range 120,000-30,000 Da were demonstrated by immunoblotting. Heavy chain 1 of inter-alpha-trypsin inhibitor was shown to associate with hyaluronate by amino acid sequence analysis of isolated hyaluronate-bound proteins. These data indicate that in vivo metabolism of inter-alpha-trypsin inhibitor takes place in pathological synovial fluid and in ovarian follicular fluid shortly before ovulation.

Alpha-Globulins↗
PubMed1998

Selective separation of trypsin from pancreatin using bioaffinity in reverse micellar system composed of a nonionic surfactant.

Selective separation of trypsin from a mixture involving many kinds of contaminating proteins, i.e., pancreatin, was achieved using trypsin inhibitor immobilized in the reverse micelles, which were composed of a nonionic surfactant, tetra-oxyethylene monodecylether. To determine the efficient operations throughout the whole separation process we examined the operating conditions, which affect the immobilization efficiency of trypsin inhibitor and also the forward and backward extractions of trypsin. Fifty percent of the recovery of trypsin from pancreatin was realized with no loss of activity of the recovered trypsin.

Animals↗