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PubMed1996

Ligation-induced acute pancreatitis increases pancreatic circulating trypsinogen activation peptides.

Ligation of the common bile-pancreatic duct induces hyperamylasemia and acute pancreatitis in rats. Pancreatic morphologic changes include edema, acinar cell damage, and mild inflammation. The pathogenesis of acute pancreatitis in this model is not understood, but may involve altered secretion and intrapancreatic activation of acinar proteases. We hypothesized that trypsinogen activation, measured by the production of plasma and pancreatic trypsinogen activation peptides (TAP), occurs early in this model. We performed the following experiments: rats were prepared with (1) bile-pancreatic ducts ligated and (2) ducts dissected but not ligated (sham). Rats were killed after 6, 24, and 48 hr. Serum amylase was measured and histologic sections were analyzed for morphologic changes. TAP was measured in both serum and pancreatic tissue homogenates using a specific polyclonal. anti-TAP antibody in an enzyme-linked immunosorbant assay. After 6, 24, and 48 hr of bile-pancreatic duct ligation, hyperamylasemia and acute morphologic changes of acute pancreatitis were observed. Evidence of acinar cell destruction was not evident until 48 hr after ligation. Levels of serum and pancreatic tissue TAP were significantly elevated at both 24 and 48 hr after ligation compared to those of sham. We conclude that increased intrapancreatic trypsinogen activation occurs early in this form of experimental acute pancreatitis and that it occurs prior to evidence of acinar cell destruction. These data and observations support the possibility that intrapancreatic protease activation contributes to the pathogenesis of ligation-induced acute pancreatitis.

Acute Disease↗
PubMed1999

Different patterns in immunoreactive anionic and cationic trypsinogen in urine and serum in human acute pancreatitis.

BACKGROUND: Acute pancreatitis (AP) results in elevated concentrations of trypsinogen (T) isoenzymes in serum. Immunoreactive anionic trypsinogen in urin (irAT/u) is elevated in AP, and has recently been proposed as a rapid diagnostic instrument and severity predictor. These results have not been confirmed by other groups, and irAT/u has not been further characterized. The concentration of immunoreactive cationic trypsinogen in urine (irCT/u) and the serum irAT/irCT ratio in AP have not been extensively examined. METHODS: Levels of irAT and irCT were studied in urine and serum from 50 AP patients and in urine from 41 non-AP patients. Severity was assessed according to the Atlanta classification. irAT/u was characterized by gel filtration. RESULTS: Gel filtration revealed only AT in the urine. Highly significant differences in irAT/u were seen between AP/non-AP (p < 0.0001) and mild/severe disease (p = 0.0012). The irAT/irCT ratio in serum changed from normal 0.8 to 1.3 in AP. CONCLUSIONS: IrAT and only traces of irCT were found in the urine in AP. IrAT/u was higher in AP than in other acute abdominal disorders (non-AP) and also higher in severe than in mild AP. IrAT in serum (irAT/s) increased proportionally more than irCT/s in AP, but did not discriminate mild from severe forms. High levels of irAT/u in some non-AP cases and a wide range in AP cases make the clinical value of the test questionable.

Acute Disease↗
PubMed1997

Stimulation with cholecystokinin leads to increased ratio between mRNA levels for anionic and cationic trypsinogen in rat pancreas.

An increased ratio between serum levels of immunoreactive anionic and cationic trypsin is a common finding in many forms of pancreatic disease. Experimental studies have shown that increased stimulation with cholecystokinin (CCK) leads to an increase in the ratio between the pancreatic content of anionic and cationic trypsin. To study whether this effect is caused by increased pancreatic synthesis of anionic trypsin in relation to cationic trypsin we studied the levels of mRNA for anionic and cationic trypsinogen in pancreatic extracts from rats exposed to increased CCK levels through chronic subcutaneous administration of CCK. Northern blot and slot blot hybridization techniques were used. The ratio between mRNAs for anionic and cationic trypsin was significantly higher in CCK-treated rats: median level, 2.04 (range, 1.33-4.08) versus a median level of 1.15 (range, 0.97-2.17) in the control group: P < 0.01. These findings support the view that chronic CCK stimulation leads to the increased synthesis of anionic trypsinogen compared to cationic trypsinogen.

Animals↗
PubMed1976

The effect of lanthanide ions on enteropeptidase-catalyzed activation of trypsinogen.

Rare earths were found to be powerful inhibitors of enteropeptidase-catalyzed (enterokinase, EC 3.4.21.9) activation of trypsinogen. Inhibition was complete at a La3+ concentration of 12.5-10(-6) M in the assay system used and still detectable at a concentration of 1.25-10(-6) M. Inhibition was observed with all lanthanides tested. No significant differences between individual metals could be established under the conditions of the inhibition assay. Increasing ionic strength decreased enzyme activity and progressively diminished the inhibitory effect of rare earth ions suggesting an electrostatic basis for the mechanism of this inhibition. La3+ did not significantly affect enteropeptidase-mediated hydrolysis of N-benzoyl-L-arginine ethyl ester. Its inhibitory effect on activation of trypsinogen by enteropeptidase, therefore, must be attributed to interaction with the zymogen rather than the enzyme. Kinetic measurements show that inhibition by rare earths is noncompetitive in nature. Binding of lanthanides to the tetraaspartyl sequence near the aminoterminus of trypsinogen may prevent this group from interacting with a critical specificity subsite on the enzyme.

Duodenum↗
PubMed1979

Studies on the activation of canine trypsinogens in vitro.

The activation of canine anionic and cationic trypsinogen by enterokinase, trypsin, thrombin, plasmin and extracts from canine granulocytes were studied in vitro. Enterokinase activates both trypsinogens about 1000 times faster than trypsin. The enterokinase-catalyzed activation is not inhibited by the main serum protease inhibitors, alpha-macroglobulin and alpha 1-antitrypsin. alpha-Macroglobulin cannot inhibit the activation of the trypsinogens by trypsin but this reaction is completely inhibited by alpha 1-antitrypsin. The results are discussed in relation to the pathogenesis of acute pancreatitis.

Animals↗
PubMed1985

The generation of lysolecithin by enterokinase in trypsinogen prophospholipase A2 lecithin mixtures, and its relevance to the pathogenesis of acute necrotising pancreatitis.

The cascade enterokinase-trypsinogen-prophospholipase A2 lecithin, generating trypsin, phospholipase A2 and lysolecithin, respectively, was studied in vitro using a novel phospholipase A2 assay. The rate of enterokinase catalysed activation of trypsinogen was maximal at 4 mmol/1 glycodeoxycholic acid; higher concentrations of bile salt progressively inhibited enterokinase activity. Net phospholipase A2 activity in reaction mixtures was critically dependent on the trypsin/prophospholipase A2 molar ratio. Lecithin hydrolysis by phospholipase A2 was dependent on the bile salt/lecithin molar ratio and was optimal at 1.25 to 1. The addition of enterokinase to lecithin and bile salt mixtures, containing trypsinogen and prophospholipase A2 at presumed pathophysiological concentrations, resulted in the generation of concentrations of lysolecithin lytic for pancreatic acinar cells within 5 min. These findings would support the concept that the entry of bile containing active enterokinase into the pancreatic duct system in vivo may in some cases be involved in the initiation of necrotising acute pancreatitis in man.

Acute Disease↗
PubMed1990

Trypsinogen activation peptides assay in the early prediction of severity of acute pancreatitis.

Trypsinogen activation can be quantified by measurement of released activation peptides (TAP assay). TAP assay in urine was performed on admission for 55 patients with acute pancreatitis. TAP concentration correlated with subsequent disease severity in 87%, whereas C-reactive protein concentration, and multifactorial scoring at 48 h, were correct in 55% and 84%. Sensitivity and specificity for TAP assay were 80% and 90%, for C-reactive protein 53% and 55%, and for multifactorial scoring at 48 h, 60% and 93%. Urine TAP assay distinguishes acute pancreatitis without trypsinogen activation from acute pancreatitis with trypsinogen activation, and helps to identify patients who will progress to the severe acute disease. Use of the assay should allow early intensive treatment of those who need it.

Acute Disease↗
PubMed1987

Differences in effect of Mn(II), Fe(III), Co(II), and Zn(II) ions on trypsinogen activation.

The influence of Mn2+, Fe3+, Co2+, and Zn2+ ions on the extent of trypsinogen activation has been determined for several ion concentrations at pH 7.4 and 36.4 degrees C. For the Mn2+ ion also the autocatalytic rate constants have been detected. The effect of Ca2+ has been reinvestigated for comparison purposes. The apparent dissociation constants of KMn2+ = 0.01 (M) and KCa2+ = 0.02 (M) have been found for the given metal ion-trypsinogen complexes. For Co2+ ion, however, only a slight effect and for Fe3+ and Zn2+ ions no significant effect could be detected on trypsinogen activation. The investigated ions are of empty, open, and completed d subshells of electrons and they are different also in their ionic size. The differences in effects of the ions are discussed on the basis of these factors.

Copper↗
PubMed1985

Activating effect of the Ile-Val dipeptide on the catalytic properties of bovine trypsinogen.

The dependence of pre-steady-state and steady-state kinetics for the trypsinogen-catalyzed hydrolysis of ZArgONp on the concentration (up to 2.0 M) of the Ile-Val effector dipeptide has been investigated at pH 8.0 and 21 +/- 0.5 degrees C. Kinetic parameters for the hydrolysis of ZArgONp catalyzed by the Ile-Val:zymogen adduct are more favorable than those observed for the free proenzyme but lower than those reported for beta-trypsin; these data indicate that the effector dipeptide induces only a partial activation of the zymogen even under saturating Ile-Val concentrations. From the dependence of kinetic parameters of proenzyme catalysis on the effector dipeptide concentration, values of the equilibrium constants for binding of Ile-Val to the free trypsinogen, to the reversible zymogen-ZArgONp complex and to the proenzyme-ZArg acyl intermediate have been obtained. Thermodynamics of binding of Ile-Val to trypsinogen, in the presence and absence of substrates and inhibitors, are indicative of the presence of different activation levels of the proenzyme, none of which is superimposable on that of beta-trypsin. On this basis, it is suggested that some of these different states could correspond to those involved in the zymogen-to-active-enzyme transition, which should be considered as a multistep process, rather than an all-or-nothing event.

Animals↗
PubMed1990

Unusual fucoidin-binding properties of chymotrypsinogen and trypsinogen.

Previous work (Jones, R. (1987) Cell Biol. Int. Reports 11, 833 and Jones et al. (1988) Development 102, 781-792) has shown that sperm proacrosin (the zymogen form of the acrosomal proteinase acrosin, EC 3.4.21.10) has the capacity to recognize and bind sulphated polysaccharides and that this property is important for the initial stages of fertilization in mammals. To investigate whether this behaviour is specific to proacrosin, a variety of other proteinases (chymotrypsinogen, trypsinogen, thrombin, elastase, plasminogen, pepsin, Streptomyces griseus proteinase and V8 proteinase from Staphylococcus aureus) were immobilized on nitrocellulose and probed with [125I]fucoidin. Only chymotrypsinogen and trypsinogen retained significant amounts of the probe with Kd values of 1.4.10(-6) M and 3.0.10(-5) M, respectively. Proteinase inhibitors were ineffective as blocking agents suggesting that enzymic activity is not involved in recognition. However, the tertiary structure of the proteins is important, since cleavage of intramolecular disulphide bonds with 2-mercaptoethanol reduced binding by 50-60%. Competition experiments with a variety of mono- and polysaccharides suggest that the number and disposition of sulphate groups is critical for interaction with basic residues on the protein. It is concluded that, like proacrosin, chymotrypsinogen and trypsinogen are bifunctional proteins.

Acrosin↗
PubMed1991

Evaluation of the kinetic parameters of the activation of trypsinogen by trypsin.

Kinetic analysis of the mechanism of trypsinogen activation by trypsin under rapid equilibrium conditions and certain relationships between the rate constants are presented. The kinetic equations are valid from the beginning of the reaction. In addition, we suggest a procedure, based on the above equations, for the evaluation of the kinetic parameters of the reaction. This procedure is applied to a set of experimental data collected during the activation of bovine trypsinogen by trypsin at 30 degrees C (pH 8.1) in 0.01 M CaCl2. In this system, the amount of active enzyme increases exponentially, as expected from an autocatalytic process. The apparent rate constant, delta, governing this increase would vary linearly with the trypsinogen concentration, [Z]0, if no Michaelis complex was detectable. However, the increase in delta with [Z]0 is clearly non-linear and fits a hyperbola (delta = k2[Z]0/(Kz + [Z]0)) well.

Animals↗
PubMed1986

Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens.

Two cDNA clones encoding two major human trypsinogen isozymes were isolated from a human pancreatic cDNA library. The deduced amino acid (aa) sequences of the two trypsinogen precursors are found to have 89% sequence homology, and have the same number of aa (247), including 15 aa for a signal peptide and 8 aa for an activation peptide. Southern blot analysis of human genomic DNA using the cloned cDNA as a probe, revealed that the human trypsinogen genes constitute a multigene family of more than ten genes.

Amino Acid Sequence↗
PubMed2004

Trypsinogen expression during the development of the exocrine pancreas in winter flounder (Pleuronectes americanus).

Histological, biochemical and molecular techniques were used to describe the functional development of the pancreas in winter flounder (Pleuronectes americanus) with specific reference to the expression of three trypsinogen genes. The pancreas was identified shortly following hatch, appearing as a compact structure situated dorsal and slightly posterior to the liver. As the larval fish approached metamorphosis, the pancreas became diffuse, spreading throughout the mesentery surrounding the stomach, the upper intestine and the pyloric caecae. Trypsin 2 expression was detected from 5 days post-hatch (dph). Two other related trypsinogen genes isolated from the pyloric caecae (Trypsin 1) and the intestine (Trypsin 3) showed contrasting results. Trypsin 1 showed very low levels of expression and only in late larval stages and metamorphosis. Trypsin 3 showed expression only after 20 dph. In order to determine tissue-specific expression of the three trypsinogen genes, the RNA from seven gastrointestinal-associated tissues was examined. Trypsin 1 and Trypsin 2 expression was most notably associated with the pyloric caecae, cardiac stomach, pyloric stomach and the rectum, although some variation in expression level between tissues was observed. Trypsin 3 expression had a narrower tissue distribution and was only associated with the pyloric caecae and the rectum. The tissue expression patterns observed here are likely due in part to the diffuse nature of the pancreas. Trypsin-like activity was evident from hatch and continued at significant levels through to at least 25 dph.

Animals↗
PubMed2001

Clinical characterization of patients with hereditary pancreatitis and mutations in the cationic trypsinogen gene.

PURPOSE: We determined the clinical manifestations of hereditary pancreatitis in nearly 30 families. PATIENTS AND METHODS: The two trypsinogen mutations N29I and R122H were identified in a group of 550 patients with chronic pancreatitis of unclear origin. The following criteria were used to characterize the severity of chronic pancreatitis (one point each): calcifications, cysts, dilation of the pancreatic duct, diabetes, hospital treatment, and operation. Stages were defined as stage 0 (no points), stage 1 (one to two points), stage 2 (three to four points), and stage 3 (more than four points). Smoking and drinking habits were also recorded. RESULTS: Six families with the N29I mutation (25 subjects with the mutation) and 21 families with the R122H mutation (76 subjects with the mutation) were identified. The median ages for the onset of disease were 11 years in N29I and 10 years in R122H patients. The severity of chronic pancreatitis and symptoms were similar for both mutations. About 26% (n = 26) of the 101 subjects carrying a mutation were asymptomatic, and 42% (n = 42) had mild disease (stage 1). Twenty-nine percent (n = 29) had moderate disease (stage 2), and only 4% (n = 4) had severe disease (stage 3). CONCLUSIONS: Symptoms of patients with the N29I or R122H trypsinogen mutation were generally similar. The majority of subjects with trypsinogen mutations had mild disease or was asymptomatic.

Adolescent↗
PubMed1999

Characterization of midgut trypsin-like enzymes and three trypsinogen cDNAs from the lesser grain borer, Rhyzopertha dominica (Coleoptera: Bostrichidae).

Protein digestion in the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), results from the action of a complex of serine proteinases present in the midgut. In this study we partially characterized trypsin-like enzyme activity against N-alpha-benzoyl-L-arginine p-nitroanilide (BApNA) in midgut preparations and cloned and sequenced three cDNAs for trypsinogen-like proteins. BApNAase activity in R. dominica midgut was significantly reduced by serine proteinase inhibitors and specific inhibitors of trypsin, whereas BApNAase activity was not sensitive to specific inhibitors of chymotrypsin or aspartic proteinases. However, trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) inhibited BApNAase activity by about 30%. BApNAase was most active in a broad pH range from about pH 7 to 9.5. The gut of R. dominica is a tubular tract approximately 2.5 mm in length. BApNAase activity was primarily located in the midgut region with about 1.5-fold more BApNAase activity in the anterior region compared to that in the posterior region. Proteinases with apparent molecular masses of 23-24 kDa that were visualized on casein zymograms following electrophoresis were inhibited by TLCK. Three cDNAs for trypsinogen-like proteins were cloned and sequenced from mRNA of R. dominica midgut. The full cDNA sequences consisted of open reading frames encoding 249, 293, and 255 amino acid residues for RdoT1, RdoT2, and RdoT3, respectively. cDNAs RdoT1, RdoT2, and RdoT3 shared 77-81% sequence identity. The three encoded trypsinogens shared 54-62% identity in their amino acid sequences and had 16-18 residues of signal peptides and 12-15 residues of activation peptides. The three predicted mature trypsin-like enzymes had molecular masses of 23.1, 28, and 23.8 kDa for RdoT1, RdoT2, and RdoT3, respectively. Typical features of these trypsin-like enzymes included the conserved N-terminal residues IVGG62-65, the catalytic amino acid triad of serine proteinase active sites (His109, Asp156, Ser257), three pairs of conserved cysteine residues for disulfide bridges, and the three residues (Asp251, Gly274, Gly284) that determine specificity in trypsin-like enzymes. In addition, RdoT2 has both a PEST-like sequence at the C-terminus and a free Cys158 near the active site, suggesting instability of this enzyme and/or sensitivity to thiol reagents. The sequences have been deposited in GenBank database (accession numbers AF130840 for RdoT1, AF130841 for RdoT2, and AF130842 for RdoT3).

Amino Acid Sequence↗
PubMed1987

Trypsinogen-trypsin transition: a molecular dynamics study of induced conformational change in the activation domain.

The trypsinogen to trypsin transition has been investigated by a stochastic boundary molecular dynamics simulation that included a major portion of the trypsin molecule and the surrounding solvent. Attention focused on the "activation domain", which crystallographic studies have shown to be ordered in trypsin and disordered in its zymogen, trypsinogen. The chain segments that form the activation domain were found to exhibit large fluctuations during the simulation of trypsin. To model a difference between trypsin and trypsinogen, the N-terminal residues Ile-16 and Val-17 were removed in the former and replaced by water molecules. As a result of the perturbation, a structural drift of 1-2 A occurred that is limited to the activation domain. Glycine residues are found to act as hinges for the displaced chain segments.

Amino Acid Sequence↗
PubMed1987

Detection of intermediate species in the refolding of bovine trypsinogen.

The mixed disulfide of bovine trypsinogen and glutathione was refolded at pH 8.6 and 4 degrees C with a mixture of 3 mM cysteine and 1 mM cystine catalyzing disulfide interchange. The folding process was monitored by analysis of quenched samples with isoelectric focusing and size-exclusion chromatography. Isoelectric focusing showed a progressive change from a pI of 5.2 for the mixed disulfide derivative to a pI of 9.3 for native trypsinogen. A number of principal intermediates were detected as a function of the refolding time. These intermediates were also separated and further characterized by size-exclusion chromatography on columns of TSK G2000 SW operated in the high-performance liquid chromatographic mode. Rechromatography of a series of sequential fractions taken from the parental peak was necessary to resolve and characterize the principal intermediates. The loss of glutathione moieties produced a partly folded structure with an apparent hydrodynamic volume (Stokes radius, Rs) of 33.9 A. These structures became compact with time, and more intermediates were detected between 33.9 and 29.2 A. Finally, a change in conformation, resembling a two-state transition, changed the molecules of Rs 29.2 to the compact structure of native trypsinogen (22.4 A). The rate of formation of the native structure was determined from the progress curves derived from isoelectric focusing and size-exclusion chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

High-level bacterial expression and 15N-alanine-labeling of bovine trypsin. Application to the study of trypsin-inhibitor complexes and trypsinogen activation by NMR spectroscopy.

We describe here the high-level expression of bovine trypsinogen in E. coli, its refolding and activation to beta-trypsin, and the selective incorporation of (15)N-labeled alanine through supplementation of the growth medium. Using this procedure, we expressed (15)N-labeled S195A trypsinogens, both on a wild-type and on a D189S background, in amounts suitable for NMR spectroscopy. 2D [(1)H-(15)N]-HSQC NMR was used to follow conformational changes upon activation of trypsinogen and formation of noncovalent complexes between S195A or S195A/D189S trypsin and protein proteinase inhibitors of different structural families and different sizes, as well as to examine the effects of introduction of the D189S mutation. Spectra of good quality were obtained for both trypsins alone and in complexes of increasing size with the proteinase inhibitors BPTI (total molecular mass 31 kDa), SBTI (total molecular mass 44 kDa), and the serpin alpha(1)-proteinase inhibitor Pittsburgh (alpha(1)PI Pittsburgh) (total molecular mass 69 kDa). Assignments of alanines 55 and 56, close to the active site histidine, and of alanine 195, present in the S195A variant used for most of the studies, were made by mutagenesis. These three alanines, together with two others, probably close to the S1 specificity pocket, were very sensitive to complex formation. In contrast, the remaining 10 alanines were invariant in chemical shift in all 3 of the noncovalent complexes formed, reflecting the conservation of structure in complexes with BPTI and SBTI known from X-ray crystal structures, but also indicating that there is no change in backbone conformation for the noncovalent complex with alpha(1)PI, for which there is no crystal structure. This was true both for S195A and for S195A/D189S trypsins. This high-level expression and labeling approach will be of great use for solution NMR studies on trypsin-serpin complexes, as well as for structural and mechanistic studies on trypsin variants.

Alanine↗