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PubMed1998

Structure of single-disulfide variants of bovine pancreatic trypsin inhibitor (BPTI) as probed by their binding to bovine beta-trypsin.

Native bovine pancreatic trypsin inhibitor (BPTI) contains three disulfide bonds: Cys5-Cys55, Cys14-Cys38 and Cys30-Cys51. Correct cysteine pairing, native structure, and full anti-proteinase activity can be restored in the process of oxidative refolding of reduced BPTI. Oxidative refolding starts with the formation of single disulfide intermediates. All 15 single-disulfide variants of BPTI (three native and 12 non-native combinations) have been expressed in Escherichia coli. In each variant the remaining four cysteine residues were replaced by alanine. Four of these variants are shown here to inhibit bovine beta-trypsin: three of them contain native and one non-native (Cys5-Cys51) disulfide. All but one (Cys5-Cys55) variant are slowly digested by the enzyme, therefore measurements were performed at pH 4.0, at which trypsin activity is low. Binding constants of these four single disulfide variants were at least two orders of magnitude lower than for native BPTI. Remarkably, in some of the variants the binding constants were found to be higher for the reduced rather than for the oxidized form of the variant. Also for the fully reduced native BPTI, determined here, the binding constant is of considerable value. Two sets of control experiments demonstrated that the binding of reduced native BPTI to trypsin is specific. In the first, mutation of Lys15 (P1 position) in the binding loop abolished binding of the reduced forms to trypsin. In the second, the binding of reduced native BPTI to anhydrotrypsin yielded the expected UV difference spectra. In general, the results obtained indicate that the inhibitor activity can be induced even in the reduced protein. This activity is not a local effect, such as the nature of residues surrounding the binding loop, but rather is induced by residual structure in the unfolded protein. This structure has been shown to consist of a set of hydrophobic residues and the data presented here indicate that reduced cysteine residues provide further stabilization of such a hydrophobic cluster. On the other hand, improper pairing of the cysteine residues in non-native single disulfide variants destabilizes the enzyme-inhibitor complex by inducing deformations of the binding loop region.

Animals↗

PubMed1994

Affinity precipitation of trypsin with soybean trypsin inhibitor linked Eudragit S-100.

Soybean trypsin inhibitor linked to Eudragit S-100 was used for the affinity precipitation of trypsin. Polymer and ligand concentrations used in conjugate preparation showed remarkable effect on the trypsin recovery. Trypsin precipitation efficiency amounted to 89% and recovery was 74%. The final purification of relatively crude commercial trypsin resulted in 1.85-fold purification. The SDS-PAGE analysis indicated significant purification. The precipitated enzyme activity was around 96% and recovered enzyme activity was 83%.

Acrylic Resins↗

PubMed1984

Comparison of human acrosin, a trypsin-like sperm proteinase, with human pancreatic trypsin: temperature stability and effect of cations.

Temperature and ion sensitivity of human acrosin (EC 3.4.21.10) was compared to that of human trypsin. With the exception of zinc, no ion tested had significant effects on either enzyme. Zinc behaved as a noncompetitive inhibitor of both enzymes, with inhibition constants of 1.8 and 1.7 mM for acrosin and trypsin respectively. Trypsin was inhibited by the chelators EDTA and EGTA, a specific effect reversed by either calcium or magnesium. EDTA inhibited acrosin in a nonspecific manner, while EGTA was without effect. Unlike acrosin from the other species, human acrosin was unaffected by calcium or the polyamines, spermine and spermidine. Acrosin was sensitive to inhibition by preincubation temperatures above 5 degrees C; trypsin, however, was stable to preincubation temperatures up to 60 degrees C. Hydrolysis of N-alpha-benzoyl-L-arginine ethyl ester was more efficiently catalyzed by trypsin (6800 cal/mol) than by acrosin (9511 cal/mol).

Acrosin↗

PubMed1981

Effects of antibodies, trypsin, and trypsin inhibitors on susceptibility of neonates to rotavirus infection.

Levels of antirotaviral secretory immunoglobulin A were measured by enzyme-linked immunosorbent assay in colostrum and milk samples collected daily for the first 5 days postpartum from 49 mothers breast-feeding their infants. The trypsin-inhibitory capacity of these lacteal secretion samples was assessed by their ability to inhibit the hydrolysis of alpha-N-benzoyl-DL-arginine-p-nitroanilide by trypsin. Stools passed by these breast-fed infants and by an additional 43 bottle-fed infants were pooled by individual and examined by electron microscopy for rotavirus. Stool trypsin levels were estimated with the gelatin hydrolysis test. Breast-fed infants were significantly less likely to become infected with rotavirus and showed significantly lower stool tryptic activity than did bottle-fed infants. Breast-fed infants who did not excrete rotavirus over the 5-day period received milk of significantly higher antirotaviral secretory immunoglobulin A or trypsin-inhibitory capacity or both than breast-fed infants who were infected with rotavirus. A case of probable maternal rotavirus infection during pregnancy, producing greatly elevated lacteal antirotaviral secretory immunoglobulin A levels lasting for 2 years, was detected. Results of this study suggest that both antibodies and trypsin inhibitors in human milk can be associated with the protection of neonates against rotavirus infection in the first 5 days of life.

Colostrum↗

PubMed1987

Binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds to bovine beta-trypsin: thermodynamic study.

The effect of pH and temperature on the association equilibrium constant (Ka) for the binding of the trypsin inhibitor from white mustard (Sinapis alba L.) seeds (MTI) to bovine beta-trypsin (EC 3.4.21.4) has been investigated. On lowering the pH from 9 to 3, values of Ka for MTI binding to bovine beta-trypsin decrease thus reflecting the acid-pK and -midpoint shifts, upon inhibitor association, of two independent ionizable groups, and of a three-proton transition, respectively. At pH 8.0, values of thermodynamic parameters for MTI binding to bovine beta-trypsin are: Ka = 4.5 X 10(8)M-1, delta G0 = -11.6 kcal/mol, and delta S0 = +53 entropy units (all at 21 degrees C); and delta H0 = +4.1 kcal/mol (temperature independent between 5 degrees C and 45 degrees C). Binding properties of MTI to bovine beta-trypsin have been analyzed in parallel with those concerning macromolecular inhibitor association to serine (pro)enzymes.

Animals↗

PubMedDate not supplied

[Trypsin-like proteinases and trypsin inhibitors in fruiting bodies of higher fungi].

The activity of trypsin-like proteinases and trypsin inhibitors was measured in fruiting bodies of various species of basidial fungi (Basidiomycetes). Fruiting bodies of all fungi contained these enzymes, with the exceptions of polypore (Coriolus versicolor (Fr.) Karst) and hedgehog fungus (Hericium erinaceus (Fr.) Quel), belonging to the families Polyporaceae and Hericiaceae, respectively, in which the enzyme activities were barely detectable. The activity of trypsin-like proteinases was the highest in fruiting bodies of Boletaceae and Agaricaceae. Fruiting bodies of all fungi contained trypsin inhibitors. The highest activity of trypsin inhibitors was detected in basidiomycetes of the families Boletaceae, Agaricaceae, and Pleurotaceae, including Boletus castanus (Fr.) Karst, orange-cap boletus (Leccinum aurantiacum (Fr.) Sing), and brown-cap boletus (Leccinum melanum (Fr.) Karst).

Basidiomycota↗

PubMed1982

Mouse plasma trypsin inhibitors. Isolation and characterization of alpha-1-antitrypsin and contrapsin, a novel trypsin inhibitor.

The trypsin-inhibiting capacity of male adult mouse plasma was several-fold greater than that of the corresponding human plasma. Two major trypsin inhibitors were isolated from mouse plasma in an apparently homogeneous state. One seems to be homologous to alpha-1-antitrypsin isolated from several mammalian species, while the other, tentatively named contrapsin, does not correspond to any of the known plasma protease inhibitors that have been well characterized in human or other animals. When human plasma was fractionated in exactly the same fashion, no inhibitor corresponding to contrapsin was detected, suggesting the absence of this inhibitor in human plasma. No cross-reactivity was observed either between contrapsin and antiserum specific for alpha-1-antitrypsin or between alpha-1-antitrypsin and antiserum specific for contrapsin. alpha-1-Antitrypsin inhibited three serine proteases tested, i.e. porcine elastase, bovine trypsin, and chymotrypsin, whereas contrapsin inhibited only trypsin, but not the other two. The two inhibitors, however, shared a number of other properties in common. They were monomeric glycoproteins having total carbohydrate content of 9.6% in alpha-1-antitrypsin (Mr = 53,000) and 15.1% in contrapsin (Mr = 55,000). They had similar amino acid and carbohydrate compositions. They inhibited bovine beta-trypsin by forming stable complexes. They showed similar sex differences in blood concentrations. Serum levels of alpha-1-antitrypsin and contrapsin in males were 5.2 +/- 0.6 and 2.8 +/- 0.2 mg/ml, respectively, whereas those in females were 3.5 +/- 0.2 and 2.1 +/- 0.4 mg/ml, respectively.

Amino Acids↗

PubMed1997

Crystal structures of bovine chymotrypsin and trypsin complexed to the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) and basic pancreatic trypsin inhibitor (BPTI): engineering of inhibitors with altered specificities.

The crystal structures of the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) complexed to bovine chymotrypsin (C-APPI) and trypsin (T-APPI) and basic pancreatic trypsin inhibitor (BPTI) bound to chymotrypsin (C-BPTI) have been solved and analyzed at 2.1 A, 1.8 A, and 2.6 A resolution, respectively. APPI and BPTI belong to the Kunitz family of inhibitors, which is characterized by a distinctive tertiary fold with three conserved disulfide bonds. At the specificity-determining site of these inhibitors (P1), residue 15(I)4 is an arginine in APPI and a lysine in BPTI, residue types that are counter to the chymotryptic hydrophobic specificity. In the chymotrypsin complexes, the Arg and Lys P1 side chains of the inhibitors adopt conformations that bend away from the bottom of the binding pocket to interact productively with elements of the binding pocket other than those observed for specificity-matched P1 side chains. The stereochemistry of the nucleophilic hydroxyl of Ser 195 in chymotrypsin relative to the scissile P1 bond of the inhibitors is identical to that observed for these groups in the trypsin-APPI complex, where Arg 15(I) is an optimal side chain for tryptic specificity. To further evaluate the diversity of sequences that can be accommodated by one of these inhibitors, APPI, we used phage display to randomly mutate residues 11, 13, 15, 17, and 19, which are major binding determinants. Inhibitors variants were selected that bound to either trypsin or chymotrypsin. As expected, trypsin specificity was principally directed by having a basic side chain at P1 (position 15); however, the P1 residues that were selected for chymotrypsin binding were His and Asn, rather than the expected large hydrophobic types. This can be rationalized by modeling these hydrophilic side chains to have similar H-bonding interactions to those observed in the structures of the described complexes. The specificity, or lack thereof, for the other individual subsites is discussed in the context of the "allowed" residues determined from a phage display mutagenesis selection experiment.

Amino Acid Sequence↗

PubMed1989

Behaviour of serum immunoreactive trypsin after serum activation by enterokinase in normal and cystic fibrosis patients. Evidence of a trypsin-alpha 1-proteinase inhibitor complex in some cystic fibrosis patients.

Serum immunoreactive trypsin (IRT) concentrations are elevated in newborn children with cystic fibrosis (CF) and subsequently fall, in most cases, to values below normal. To evaluate the molecular form(s) of IRT present in serum, we have performed serum activation by enterokinase and have measured serum IRT before and after activation. This approach is based on the postulate that enterokinase converts trypsinogen into trypsin, and this trypsin would then be mainly trapped by alpha 2-macroglobulin, thus escaping the assay. This assumption was confirmed in the 28 controls studied, where the mean percentage (+/- S.D.) of IRT recovery after serum activation was 13.7 +/- 2.9. Previous inhibition of alpha 2-macroglobulin by methylamine raised the recovery over 85%, confirming that most of the serum IRT present in controls was in the form of trypsinogen. Identical results were obtained in the serum of 10 obligate heterozygotes and in 57 out of 80 CF patients. In 23 CF patients the mean percentage of IRT recovery after serum activation was 41.6 +/- 17.6. Gel-filtration studies were performed on the sera of the CF patients showing an abnormal increase in the IRT recovery after serum activation. We could demonstrate that IRT was distributed in two fractions: one eluted with the Mr 25,000 protein as usually found in controls and other CF sera, and the other eluted with the Mr 75,000 protein corresponding to a complex of trypsin with alpha 1-proteinase inhibitor. These results show that, in these sera, active trypsin has been directly released in blood. These findings suggest that in some patients with CF, subclinical attacks of acute pancreatitis may occur.

Chromatography, Gel↗

PubMed1979

Quantitative assessment of the amount and the activity of trypsin associated with trypsinized cells.

The present investigation has demonstrated that when cell layers are trypsinized with 125I-trypsin and washed under strictly standardized conditions, the amount of trypsin which remains associated with the cell suspension can be accurately determined. This amount is more than 10 times greater than would be expected from dilution only and is dependent on the density of the cell layers. On plating of the cells, less than 10% of the carry-over trypsin remains associated with the cells and suggestive evidence for a mainly intracellular localization was obtained with a quantitative immunoperoxidase technique. This carry-over trypsin was further shown to maintain proteolytic activity by its ability to remove significant amounts of macromolecular material from a cell layer prelabeled with 3H-Leucine.

Cell Count↗

PubMed2002

Crystal structure and nucleotide sequence of an anionic trypsin from chum salmon (Oncorhynchus keta) in comparison with Atlantic salmon (Salmo salar) and bovine trypsin.

The nucleotide sequence and crystal structure of chum salmon trypsin (CST) are now reported. The cDNA isolated from the pyloric caeca of chum salmon encodes 222 amino acid residues, the same number of residues as the anionic Atlantic salmon trypsin (AST), but one residue less than bovine beta-trypsin (BT). The net charge on CST determined from the sum of all charged amino acid side-chains is -3. There are 79 sequence differences between CST and BT, but only seven sequence differences between CST and AST. Anionic CST isolated from pyloric caeca has also been purified and crystallized; the structure of the CST-benzamidine complex has been determined to 1.8A resolution. The overall tertiary structure of CST is similar to that of AST and BT, but some differences are observed among the three trypsins. The most striking difference is at the C terminus of CST, where the expected last two residues are absent. The absence of these residues likely increases the flexibility of CST by the loss of important interactions between the N and C-terminal domains. Similarly, the lack of Tyr151 in CST (when compared with BT) allows more space for Gln192 in the active site thereby increasing substrate accessibility to the binding pocket. Lys152 in CST also adopts the important role of stabilizing the loop from residue 142 to 153. These observations on CST provide a complementary view of a second cold-adapted trypsin, which in comparison with the structures of AST and BT, suggest a structural basis for differences in enzymatic activity between enzymes from cold-adapted species and mammals.

Animals↗

PubMed2000

Anionic trypsin from chum salmon: activity with p-amidinophenyl ester and comparison with bovine and Streptomyces griseus trypsins.

An anionic trypsin from pyloric caeca of chum salmon (Oncorhynchus keta) was purified by ammonium sulfate and acetone fractionation followed by affinity chromatography, gel-filtration, and DEAE-anion exchange chromatography. The apparent molecular mass was about 24 kDa as determined by SDS-PAGE. The anionic chum salmon trypsin was moderately active toward esterase substrates such as tosyl-L-arginine methyl ester and tosyl-L-lysine methyl ester. Its amidase activity for benzoyl-L-arginine p-nitroanilide was comparative to those of bovine and Streptomyces griseus trypsins. Kinetic characteristics of anionic chum salmon, bovine, and Streptomyces griseus trypsins toward inverse substrate (p-amidinophenyl ester) were compared. Inverse substrate behaved as a specific substrate for anionic chum salmon trypsin with specific binding, efficient acylation, and relatively slow deacylation.

Amides↗

PubMed1988

Stimulation of high-affinity GTPase by trypsin and trypsin-like proteinases in membranes of human platelets.

The influence of various proteinases on GTP hydrolysis was studied in membranes of human platelets. Of the proteinases examined, trypsin, acrosin and a recently described trypsin-like proteinase from bovine sperm, but not chymotrypsin, increased GTP hydrolysis. Similar to what was described previously for hormone-like agents, the stimulation of GTP hydrolysis by the proteinases was only observed at low GTP concentrations, with apparent Km values of 0.2-0.3 microM-GTP. Stimulation of the high-affinity GTPase by the proteinases occurred without apparent lag phase and was constant over a long period of incubation. The proteinase inhibitors leupeptin and soya-bean trypsin inhibitor blocked the stimulation of GTP hydrolysis, but did not reverse the effect of the proteinases. Treatment of platelet membranes with N-ethylmaleimide, which eliminates Gi-protein (inhibitory guanine-nucleotide-binding protein)-related GTPase stimulation by adrenaline, decreased stimulation of GTP hydrolysis by the proteinases only partially. Activation of GTP hydrolysis by the proteinases was partially additive with that caused by adrenaline, whereas thrombin stimulation was not increased further. The data indicate that, similarly to the proteinase thrombin, trypsin and trypsin-like proteinases can activate GTP-hydrolysing protein(s) that exhibit high affinity for GTP in platelet membranes. It is suggested that the proteinases interact in platelet membranes with a receptor site similar to that used by thrombin and that the observed GTPase stimulation is a reflection of a proteinase-receptor interaction with a guanine-nucleotide-binding regulatory protein.

Blood Platelets↗

PubMed1993

Bovine pancreatic trypsin inhibitor-trypsin complex as a detection system for recombinant proteins.

Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10(-14) and 1.1 x 10(-13) M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.

Animals↗

PubMed2002

Crystal structure of bovine trypsin and wheat germ trypsin inhibitor (I-2b) complex (2:1) at 2.3 a resolution.

The Bowman-Birk trypsin inhibitor (BBI) from wheat germ (I-2b) consists of 123 amino acid residues with two inhibitory loops. The crystal structure of a bovine trypsin-wheat germ trypsin inhibitor (I-2b) complex (2:1) has been determined at 2.3 A resolution to a final R-factor of 0.177. A distance of 37.2 A between the contiguous contact loops allows them to bind and inhibit two trypsin molecules simultaneously and independently. Each domain shares the same overall fold with 8 kDa BBIs. The five disulfide bridges in each domain are a subset of seven disulfide bridges in the 8 kDa BBIs. I-2b consists of ten beta-strands and the loops connecting these strands but it lacks alpha-helices. The conformations of the contiguous contact loops of I-2b are in a heart-like structure. The reactive sites in both domains, Arg 17 and Lys 76, are located on the loop connecting anti-parallel beta-strands, beta 1/beta 2 and beta 6/beta 7. Strands beta 1 and beta 6 are in direct contact with trypsin molecules and form stable triple stranded beta-sheet structures via hydrogen bonds.

Amino Acid Sequence↗

PubMed1988

Thrombin-like inhibitory action of trypsin and trypsin-like proteases on human platelet adenylate cyclase.

The effects of trypsin, acrosin and a recently described trypsin-like protease from bovine sperm were studied on adenylate cyclase activity in membranes of human platelets. These proteases caused an immediate decrease in adenylate cyclase activity, which was independent of the platelet membrane concentration used and which was constant for up to 20 min of incubation at 25 degrees C. When the incubation was prolonged, the proteases eliminated their own inhibitory action as well as that of the inhibitory hormone epinephrine. The adenylate cyclase inhibition caused by the proteases was strictly dependent on the presence of GTP (EC50 approximately 0.1 microM), whereas in the absence of GTP only minor changes in enzyme activity were observed at the conditions and protease concentrations used. Maximal inhibition caused by the proteases was between 40% and 60%. Half-maximal inhibition by the purified proteases trypsin and acrosin was observed at about 30 ng/ml and 2 micrograms/ml respectively. Inhibition of platelet adenylate cyclase by the proteases was partially additive with that caused by epinephrine, while with thrombin no additivity was observed. The serine protease inhibitor leupeptin blocked the actions of the proteases when added simultaneously with the enzymes, but was ineffective when added later on. Treatment of platelet membranes with the alkylating N-ethylmaleimide at low concentrations and Mn2+ ions (greater than or equal to 1 mM), both agents known to abolish inhibition of adenylate cyclase via the inhibitory guanine-nucleotide-binding protein Gi, eliminated the inhibitory action of the proteases. The data indicate that trypsin and trypsin-like proteases have two opposite effects on the platelet adenylate cyclase system, the well-documented elimination of Gi action and, as shown here, an immediate activation of Gi with subsequent adenylate cyclase inhibition. The data are consistent with the hypothesis that the activation of Gi caused by the proteases is due to an interaction of the proteases with specific cell-surface receptor sites in a manner similar to thrombin.

Acrosin↗

PubMed1981

Evidence for two mechanisms of dengue virus infection of adherent human monocytes: trypsin-sensitive virus receptors and trypsin-resistant immune complex receptors.

Trypsin treatment of adherent human monocytes greatly reduced or eliminated the ability of these cells to support dengue virus replication. However, addition of dilute (nonneutralizing) antibody to the inoculum and the culture medium resulted in viral yields similar to those from monocytes not treated with trypsin. These results suggested that viral entry was facilitated by phagocytosis of immune complexes via Fc receptors on the monocytes. This concept was tested by (i) pretreating monocytes with aggregated gamma globulin, which resulted in a 40-fold reduction of viral yields after infection with dilute antibody-virus complexes and (ii) forming an immune complex with virus, antivirus F(ab')2 fragments, and rabbit anti-human Fab. Whereas F(ab')2 fragments alone would not enhance virus replication in trypsin-treated monocytes, the immune complex containing a rabbit Fc piece did increase the yield of dengue virus. These results suggest that dengue virus can infect a cultured monocyte in two ways: (i) through a viral receptor that is trypsin sensitive or (ii) through an Fc receptor that is not trypsin sensitive.

Antibodies, Viral↗

PubMed1987

Molecular size distribution of immunoreactive trypsin and renal tubular dysfunction: role in trypsin plasma-urine transfer.

In order to investigate the role of circulating free trypsinogen and renal tubular dysfunction in affecting trypsin plasma-urine transfer, serum immunoreactive trypsin (IRT), its urinary output, IRT molecular size distribution, filtrable immunoreactive trypsin, gamma-glutamyltransferase and alpha-glucosidase outputs were studied in 6 control subjects, 9 patients with pancreatic cancer and 15 with chronic pancreatitis. The majority of immunoreactivity was always eluted at a molecular weight of about 24,000 and might therefore be considered as free trypsinogen. Variable amounts of IRT at higher molecular weights, possibly represented by trypsin-inhibitor complexes, were also detected. Increasing IRT levels were generally accounted for by free trypsinogen, regardless of the nature of the disease. Unlike serum free trypsinogen levels, renal tubular damage, evaluated by means of the excretion of two high-molecular weight urinary enzymes, seems to play a prominent role in explaining trypsin plasma-urine transfer.

Adolescent↗