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PubMed1981

Inhibition of alpha 2-macroglobulin-bound trypsin by soybean trypsin inhibitor.

Soybean trypsin inhibitor, a protein of Mr = 20,000, has been used to assess the degree of inaccessibility of porcine trypsin within the alpha 2-macroglobulin-trypsin complex. The interaction between alpha 2-macroglobulin-bound trypsin and the inhibitor was demonstrated by affinity chromatography and trypsin inhibition. Whereas the free trypsin-inhibitor association is very fast (k = 1.2 X 10(7) M-1 s-1), the reaction between complexed trypsin and inhibitor takes 10 h to reach equilibrium. In addition, alpha 2-macroglobulin reduces, by several orders of magnitude, the affinity of trypsin for the inhibitor. Only one of the two trypsin molecules of the ternary (trypsin)2-alpha 2-macroglobulin complex is readily accessible to soybean inhibitor. It is postulated that the recently discovered proximity of the alpha 2-macroglobulin binding sites (Pochon, F., Favaudon, V., Tourbez-Perrin, M., and Bieth, J. (1981) J. Biol. Chem. 256, 547-550) accounts for this behavior. In the light of these results it is concluded that the proteinase binding sites are localized on the alpha 2-macroglobulin surface and that the two subunits of this protein are either not identical or not symmetrically arranged.

Animals↗
PubMed2003

Activities of anionic and cationic trypsins in the temperature range from 5 to 37 degrees C. Mutant anionic trypsins as a model of cold-adapted (psychrophilic) enzymes.

Temperature dependences of kinetic constants (k(cat) and K(m)) were studied for enzymatic hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-arginine-p-nitroanilide and N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-lysine-p-nitroanilide by bovine cationic and rat anionic (wild-type and mutant) trypsins. The findings were compared with the corresponding literature data for hydrolysis of N-benzoyl-DL-arginine-p-nitroanilide by bovine cationic trypsin and natural trypsins of cold-adapted fishes. The anionic and cationic trypsins were found to differ in organization of the S(1)-substrate-binding pocket. The difference in the binding of lysine and arginine residues to this site (S(1)) was also displayed by opposite temperature dependences of hydrolysis constants for the corresponding substrates by the anionic and cationic trypsins. The data suggest that the effect of any factor on the binding of substrates (the K(m) value) to the anionic and cationic trypsins and on the catalytic activity k(cat) should be compared only with the corresponding data for the natural enzyme of the same type. Mutants of rat anionic trypsin at residues K188 or Y228 were prepared by site-directed mutagenesis as approximate models of natural psychrophilic trypsins. Substitution of the charged lysine residue in position 188 by hydrophobic phenylalanine residue shifted the pH optimum of the resulting mutant trypsin K188F from 8.0 to 9.0-10.0, similarly to the case of some natural psychrophilic trypsins, and also 1.5-fold increased its catalytic activity at low temperatures as compared to the wild-type enzyme.

Animals↗
PubMed1975

Kinetics of binding of bovine trypsin-killikrein inhibitor (K unitz) in which the reactive-site peptide bond Lys-15--Ala-16 is cleaved, to alpha-chymotrypsin and beta-trypsin.

Equilibrium measurements of the binding of reactive-site-cleaved (modified) bovine trypsin-kallikrein inhibitor (Kunitz) to alpha-chymotrypsin and beta-trypsin show a stoichiometric 1:1 association with high binding constants. At least in the case of chymotrypsin much evidence is presented that the reaction with modified inhibitor leads to the same complex as the reaction with virgin inhibitor does. The association rate constant of modified inhibitor with chymotrypsin at pH 7, 22.5 degrees C is 15.8 M-1 S-1. This is about 2 x 10(4) times slower than the binding of virgin inhibitor to that enzyme. In the analogous reaction of modified inhibitor with beta-trypsin, however, the association rate constant (1.2 x 10(4) M-1 s-1 at pH 6.9, 22.5 degrees C) is of about the same order of magnitude as it is in the reaction of virgin inhibitor and trypsin. These and analogous phenomena observed in the reactions of virgin and modified soybean trypsin inhibitor (Kunitz) with alpha-chymotrypsin and beta-trypsin suggest that the specificity of both inhibitors to trypsin is strongly reflected in the association rate constants of the modified forms. The dissociation rate constants of the complexes of trypsin-kallikrein inhibitor with chymotrypsin or with trypsin towards the modified inhibitor are estimated to be unmeasurably slow (half-life times of 45 or 1.5 x 10(4) years, respectively).

Alanine↗
PubMed2005

Oral trypsin inhibitor can improve reflux esophagitis after distal gastrectomy concomitant with decreased trypsin activity.

BACKGROUND: The pathogenesis of reflux esophagitis is not well understood and remains controversial. Distal gastrectomy serves as a model to assess the role of duodenal reflux with low gastric acidity in the development of reflux esophagitis. We investigated the clinical usefulness and antitrypsin activity after treatment with a trypsin inhibitor, camostat mesilate, against the reflux esophagitis after distal gastrectomy reconstructed with Billroth-I anastomosis. METHODS: Twenty-eight patients with gastroesophageal reflux disease after distal gastrectomy were prescreened according to esophageal pH level and trypsin activity, and consequently 11 patients were enrolled in the present clinical study. Esophageal and duodenal washings were aspirated for the evaluation of the pretreatment trypsin activity. Then 100 mg of camostat mesilate was administered orally. At 30 and 120 minutes after the administration, duodenal washings were aspirated for the evaluation of posttreatment trypsin activity. Thereafter, 300 mg of camostat mesilate was administered orally 3 times daily for a 4-week period. On the 28th day of administration, the grade of reflux esophagitis (Los Angeles classification) was re-evaluated under endoscopy and the esophageal washings were aspirated for the evaluation of trypsin activity. RESULTS: The trypsin activities in the duodenum both at 30 and 120 minutes after oral ingestion of camostat mesilate were decreased significantly in comparison with those in the pretreatment period in each patient (P<.001). In 6 of 7 patients with detectable trypsin activity in the esophagus, the activities after the 28th day of treatment were lower than those in the pretreatment period, and the symptoms were milder than those before treatment (P<.05). Furthermore, endoscopic evaluation showed that 40% of patients were grade B, C, and D after treatment (28th day), whereas 70% of patients were grade B, C, and D before the treatment. CONCLUSIONS: Oral administration of trypsin inhibitor can improve reflux esophagitis after distal gastrectomy concomitant with decreased trypsin activity.

Administration, Oral↗
PubMed1983

Influence of inactivation of trypsin on immunoreactivity and serum immunoreactive trypsin concentration measured by radioimmunoassay.

The influences of various active site-specific reagents of trypsin and protease inhibitors on the immunoreactivity of trypsin and serum trypsin concentration have been studied by radioimmunoassay (RIA). The RIA using inactivated 125I-trypsin as tracer showed lower Bo/T than the RIA using active 125I-trypsin, but the coefficient of variance of the former was smaller than that of the latter. Normal serum trypsin concentrations were 26.12-36.38 ng/ml with the RIA using inactivated 125I-trypsin as antigen tracer, and 201.15 ng/ml with the RIA using active 125I-trypsin as tracer. The recovery experiment showed that the difference was due to the interaction of serum protease inhibitors and labeled active trypsin.

Cross Reactions↗
PubMed1983

Trypsin inhibitors from Ascaris: the reactive P1 site of the inhibitors (a correction) and location of the inhibitors and host trypsin in cross-sections of Ascaris.

Ascaris trypsin inhibitors 1, 2, and 3 have arginine at their reactive P1 site. This corrects an earlier report that lysine is the reactive P1 site residue in Ascaris trypsin inhibitor 1 (Peanasky et al., 1974, Bayer Symposium V: Proteinase Inhibitors, pp. 649-666). The present work illustrates that the residue modification method of Fritz et al. (1969, Z. Physiol. Chem., 350, 933-944) may not be reliably interpreted when trypsin inhibitors have an unusually high lysine content (greater than 12% of the molecular weight of the inhibitor). Thus the following procedure is recommended: treat the inhibitor with maleic anhydride first and second with butanedione reagent; then remove the maleyl groups in an acid environment and determine the activity of the inhibitor. Immunoperoxidase staining shows that antibody to Ascaris trypsin inhibitor 1 binds to body wall muscle, intestine, eggs and sperm in cross-sections of Ascaris. Antibody to TLCK-porcine trypsin binds to the same tissues and at the same sites as the antibody to Ascaris trypsin inhibitor 1. This is the first demonstration that a protein that originated in the host has been found in the parasite, Ascaris. Analyses of homogenates and of extracts of separated tissues always show an excess of free trypsin inhibitor and no evidence of active trypsin. The host protein is present inside the parasite, probably as the trypsin-inhibitor complex.

Animals↗
PubMed1993

Tyrosine 151 is part of the substrate activation binding site of bovine trypsin. Identification by covalent labeling with p-diazoniumbenzamidine and kinetic characterization of Tyr-151-(p-benzamidino)-azo-beta-trypsin.

Identification of the substrate activation site of beta-trypsin by a 1:1 reaction with p-diazoniumbenzamidine chloride was confirmed by spectral analysis. Proteolysis of Cm-p-benzamidino-azo-beta-trypsin provided peptides containing modified tyrosine residues. The major product, Ser-146 to Lys-156, which corresponded to labeling at Tyr-151, was recovered in 35% yield, and its structure was demonstrated by amino acid analysis, Edman degradation, and mass spectrometry. Yields of labeled Tyr-151, Tyr-39, and Tyr-172, identified by peptide analysis, were in the proportion of 100:7:3. Tyr-151-(p-benzamidino)-azo-beta-trypsin is permanently activated, but can be further activated by substrates. Values of kcat, Ks', and kcat' vary from two to three times the equivalent values for trypsin. Berenil (4,4'-diazoamino-bis-benzamidine), a parabolic competitive inhibitor of beta-trypsin, was a hyperbolic competitive inhibitor of azo-beta-trypsin. Thus, Tyr-151, part of subsite S'2, affects the catalytic process and, when modified covalently, permanently activates trypsin. Equilibrium binding with berenil supported the kinetic data obtained with substrates. This permits the integration of protein modification, kinetics, equilibrium binding, and crystallographic data to demonstrate a fine interaction between subsites S1-S3 and S'2 in trypsin and azo-beta-trypsin, resulting in subtle structural changes when the native enzyme is covalently modified at Tyr-151.

Amino Acid Sequence↗
PubMed1975

The location of the calcium ion binding site in bovine alpha-trypsin and beta-trypsin using lanthanide ion probes.

The effect of Gd3+ on the nuclear magnetic resonance (NMR) relaxation rates, T1m-1 and T2m-1, of inhibitor protons in metal-inhibitor-trypsin ternary complexes has been measured. The Solomon-Bloembergen equations have been used to calculate distances of 10.0 +/- 0.5, 8.8 +/- 0.5, and 9.5 +/- 0.5 A between the metal ion and the methyl and ortho protons of p-toluamidine, and the methyl protons of acetamidine, respectively. Essentially the same results are obtained for both alpha-trypsin and beta-trypsin. Binding constants of 3.3 x 10(3) and 4.1 x 10(3) M-1 for the association of Gd(III) with alpha-trypsin and beta-trypsin, respectively, in the presence of p-toluamidine at pH 6.0 have been obtained by equilibrium dialysis. Calcium binding constants of 260 and 3700 M-1 at pH 6.0 and 8.0, respectively, with beta-trypsin have also been obtained. Calcium ion and gadolinium ion compete for the same site on the protein. Calcium has been shown to protect alpha-trypsin from further autolytic degradation to psi-trypsin. From examination of the crystal structure of the enzyme we propose that the calcium ion binding site of bovine trypsin is comprised of the side chains of Asp-194 and Ser-190 (based on the chymotrypsin sequence numbering system). This seems to be the only site which is comprised of at least one carboxyl group; which fits our distance requirements and which is conisistent with other chemical data.

Animals↗
PubMed1974

Affinity chromatography of bovine trypsin. A rapid separation of bovine alpha- and beta-trypsin.

Affinity adsorbents for bovine trypsin were prepared by covalently coupling p-(p'-amino-phenoxypropoxy)benzamidine to cellulose and to agarose. Trypsin binds to both adsorbents at pH6-8 and is released at low pH values or in the presence of n-butylamine hydrochloride. Pure beta-trypsin may be eluted from crude trypsin bound at pH8.0 to the cellulose adsorbent by stepwise elution with an acetate buffer, pH5.0. Both alpha- and beta-trypsin may be isolated by chromatography of crude trypsin on the agarose derivative in an acetate buffer, pH4.0. These two methods for purifying the trypsin are specific to the particular adsorbents. They are rapid and convenient in use. Both methods leave a mixture of the two enzymes bound to the adsorbent and release occurs only at low pH values. The effects of pH, composition and ionic strength of buffer and other variables on both purification methods are described. Affinity adsorbents of soya-bean trypsin inhibitor and of N-alpha-(N'-methyl-N'-sulphanilyl) sulphanilylagmatine bound to agarose were prepared, but were found to be of limited usefulness in the purification of trypsin.

Amidines↗
PubMed1984

The two alpha 2-macroglobulin-bound trypsin molecules have different affinities for the basic pancreatic trypsin inhibitor.

We have investigated the enzymatic properties of alpha 2-macroglobulin-bound porcine trypsin using a substrate: Z-Gly-Gly-Arg-p-nitroanilide and two inhibitors: p-aminobenzamidine and basic pancreatic trypsin inhibitor. The ternary alpha 2-macroglobulin-(trypsin)2 complex behaves like a mixture of two enzymes which bind basic pancreatic trypsin inhibitor with widely different affinities (Ki = 0.11 microM and 23 microM). About one-half of the trypsin molecules of the ternary complex are covalently bound to alpha 2-macroglobulin. Preparation of the complex in the presence of hydroxylamine prevents covalent bond formation, but the two trypsins of this artificial complex still exhibit large differences in affinity for basic pancreatic trypsin inhibitor. The trypsin molecules of the ternary complex also exhibit small differences in their affinity for Z-Gly-Gly-Arg-p-nitroanilide and p-aminobenzamidine.

Benzamidines↗
PubMedDate not supplied

Binding of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to bovine alpha-chymotrypsin and bovine beta-trypsin. A thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C(p), F-T(p) and F-T(t), respectively) to bovine alpha-chymotrypsin (alpha-chymotrypsin) and bovine beta-trypsin (beta-trypsin) has been investigated. On the basis of Ka values, the proteinase inhibitor affinity can be arranged as follows: alpha-chymotrypsin: BBI approximately beta-trypsin:BBI approximately beta-trypsin:F-T(t) approximately beta-trypsin:F-T(p) much greater than alpha-chymotrypsin:F-C(p). F-C(p), F-T(p) and F-T(t) do not inhibit beta-trypsin and alpha-chymotrypsin action, respectively. On lowering the pH from 9.5 to 4.5, values of Ka for BBI, F-C(p), F-T(p) and/or F-T(t) binding to alpha-chymotrypsin and beta-trypsin decrease, thus reflecting the acid-pK shift of the invariant His57 catalytic residue from 7.0, in the free enzymes, to 5.2, in the proteinase:inhibitor complexes. Considering the known molecular models, the observed binding behaviour of BBI, F-C(p), F-T(p) and F-T(t) was related to the inferred stereochemistry of the proteinase:inhibitor contact regions.

Animals↗
PubMed1979

Human inter-alpha-trypsin inhibitor. Limited proteolysis by trypsin, plasmin, kallikrein and granulocytic elastase and inhibitory properties of the cleavage products.

The acid-labile inter-alpha-trypsin inhibitor is cleaved enzymatically in vivo, liberating a smaller acid-stable inhibitor. The molar ratio of native inhibitor to this smaller inhibitor in plasma is significantly changed in some severe cases of inflammation and kidney injury. To clarify this observation on a molecular basis, the action of four different types of proteinases (trypsin, plasmin, kallikrein and granulocyte elastase) on the inter-alpha-trypsin inhibitor was studied. The initial rate of cleavage of the inter-alpha-trypsin inhibitor by a 1.3-fold molar excess of proteinase over inhibitor was found to be 4375 nM x min-1 with granulocyte elastase, 860 nM x min-1 with trypsin, 67 nM x min-1 with plasmin, and 0.3 nM X min-1 with kallikrein. Obviously, of the enzymes studied so far, the granulocyte elastase known to be released during severe inflammatory processes is by far the most potent proteinase in the transformation of the inter-alpha-trypsin inhibitor. The inter-alpha-trypsin inhibitor and its cleavage products inhibit bovine trypsin very strongly (Ki = 10(-9)--10(-11) M), porcine plasmin much less strongly, human plasmin very weakly and pancreatic kallikrein practically not at all.

Alpha-Globulins↗
PubMed1980

Serum immunoreactive trypsin and trypsin inhibitors during acute pancreatitis.

Serum trypsin, total trypsin-inhibitor capacity, alpha-1-antitrypsin and alpha-2-macroglobulin were analyzed daily in ten patients hospitalized as a result of acute pancreatitis. Markedly raised serum trypsin concentrations were found in all patients. Alpha-1-antitrypsin and the trypsin-inhibitor capacity were also significantly increased as compared to post-illness values, but alpha-2-macroglobulin tended to decrease during acute pancreatitis. The post-illness values of all these parameters were in the normal range. It is concluded that a deficiency of alpha-1-antitrypsin, trypsin-inhibitor capacity of alpha-2-macroglobulin is not present in patients with acute pancreatitis which could render the pancreas more vulnerable to its own proteases. During acute pancreatitis trypsin is released into the circulation, but it is effectively inactivated by serum protease inhibitors, mainly alpha-1-antitrypsin, which can be seen as an increased trypsin-inhibitor capacity in these patients. The use of protease inhibitors in the treatment of acute pancreatitis seems to be unnecessary against this background. Increased serum trypsin values can be used when confirming the diagnosis of acute pancreatitis.

Acute Disease↗
PubMed1993

Anti-inflammatory agents inhibit leukocyte accumulation and vascular leakage induced by trypsin and trypsin-digested serum in hamster cheek pouch.

In the present study we investigated the effect of nordihydroguaiaretic acid (NDGA), indomethacin, and cortisone on trypsin-induced acute inflammation in the hamster cheek pouch. Permeability changes, evaluated by fluorescence microscopy after injection of FITC-dextran (MW 150,000), induced by trypsin (2.5 microM) and trypsinated serum (2.5 microM) were significantly suppressed by pretreatment with NDGA (20 mg/kg) and indomethacin (20 mg/kg). Pretreatment with cortisone (40 mg/kg) reduced the permeability changes induced by trypsinated serum but had no significant effect on trypsin-induced leakages. Accumulation of polymorphonuclear leukocytes, as calculated by a whole tissue histological technique, induced by trypsin or trypsinated serum, was significantly reduced by pretreatment with cortisone, NDGA, or indomethacin. These results indicate a role of both cyclooxygenase and lipoxygenase products in trypsin-induced acute inflammation in the hamster cheek pouch.

Animals↗
PubMed1980

Serum trypsin concentration and pancreatic trypsin secretion in insulin-dependent diabetes mellitus.

Fasting serum trypsin concentration and pancreatic trypsin output, stimulated by secretin and cholecystokinin-pancreozymin, were measured in 18 patients with insulin-dependent diabetes, to assess a possible correlation between these two indices of exocrine pancreatic function. Serum trypsin concentration was subnormal in 13, and pancreatic trypsin output was decreased in 14 patients, but there was no significant correlation between the two measurements. There was no correlation between serum trypsin and residual beta cell function measured by plasma C-peptide immunoreactivity (CPR). After an interval of four years serum trypsin measurements were repeated in 11 subjects. All individual trypsin levels were lower than the previous results, the difference being highly significant (t = 9.0; p < 0.001). Serum trypsin concentration therefore represents a qualitative index of reduced exocrine pancreatic function in diabetes, but has no value in quantitating the degree of deficiency.

Adolescent↗
PubMed2002

Expression and functional analysis of rat P23, a gut hormone-inducible isoform of trypsin, reveals its resistance to proteinaceous trypsin inhibitors.

Rat P23 is an isoform of trypsin (ogens) synthesized by rat acinar cells. Expression of P23 is stimulated strongly by caerulein, an analogue of cholecystokinin (CCK). However, the physiological relevance of rat P23 in healthy and pathological conditions such as caerulein-induced pancreatitis is largely unknown. Using recombinant P23 trypsinogen and reconstitution analysis of zymogen autoactivation, unique inhibitor-resistance characteristics of P23 were elucidated. P23 cDNA was expressed in Escherichia coli periplasm, yielding recombinant P23 trypsinogen. Autoactivation of zymogen granule contents from caerulein-induced rat pancreas was also studied. Activation kinetics of P23 by enterokinase was similar to those of rat anionic trypsinogen, which is a major isoform of trypsinogen. Interestingly, rat pancreatic secretory trypsin inhibitor (PSTI), which protects against deleterious activation of trypsinogens in zymogen granules, failed to inhibit P23 trypsin even with four-fold molar excess, at which concentration it effectively inhibited rat anionic trypsin to almost 100%. P23 trypsin also showed marked resistance to proteinaceous trypsin inhibitors such as soybean trypsin inhibitor and aprotinin. P23 trypsin activated by enterokinase dramatically accelerated the cascade of autoactivation of anionic trypsinogen even in the presence of PSTI. Taken together with a previous observation that P23 is specifically upregulated 14-fold by 24-h caerulein infusion, these results suggest that elevated levels of P23 should be taken into consideration in the mechanism of trypsinogens within the pancreas in pathological conditions.

Amino Acid Sequence↗