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PubMed1986

Age-related alterations of immunoreactive pancreatic cationic trypsinogen in sera from cystic fibrosis patients with and without pancreatic insufficiency.

Serum immunoreactive cationic trypsinogen levels were determined in 99 control subjects and 381 cystic fibrosis (CF) patients. To evaluate the status of the exocrine pancreas all CF patients had previously undergone fecal fat balance studies and/or pancreatic stimulation tests. Three hundred fourteen CF patients had fat malabsorption and/or had inadequate pancreatic enzyme secretion (pancreatic insufficiency) requiring oral pancreatic enzyme supplements with meals. Sixty-seven CF patients did not have fat malabsorption and/or had adequate enzyme secretion (pancreatic sufficiency) and were not receiving pancreatic enzyme supplements with meals. Mean serum trypsinogen in 99 control subjects was 31.4 +/- 14.8 micrograms/liter (+/- 2 SD) and levels did not vary with age or sex. In CF infants (less than 2 yr) with pancreatic insufficiency, mean serum trypsinogen was significantly above the non-CF values (p less than 0.001). Ninety-one percent of the CF infants had elevated levels. Serum trypsinogen values in the pancreatic insufficient group declined steeply up to 5 years, reaching subnormal values by age 6. An equation was developed which described these age-related changes very accurately. Only six CF patients with pancreatic insufficiency had serum trypsinogen levels above the 95% confidence limits of this equation. In contrast, there was no age related decline in serum trypsinogen among the CF group with pancreatic sufficiency. Under 7 yr, serum trypsinogen failed to distinguish the two groups. In those over 7 yr of age, however, serum trypsinogen was significantly higher than the CF group with pancreatic insufficiency (p less than 0.001), and 93% had values within or above the control range.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed2001

The ratio of trypsin-2-alpha(1)-antitrypsin to trypsinogen-1 discriminates biliary and alcohol-induced acute pancreatitis.

BACKGROUND: Rapid determination of the etiology of acute pancreatitis (AP) enables institution of appropriate treatment. We evaluated the ability of trypsinogen-1, trypsinogen-2, trypsin-1-alpha(1)-antitrypsin (AAT), and trypsin-2-AAT in serum to identify the etiology of AP. METHODS: The study consisted of 67 consecutive patients with AP admitted to Helsinki University Central Hospital. Forty-two had alcohol-induced AP, 16 had biliary AP, and 9 had unexplained etiology. Serum samples were drawn within 12 h after admission. Trypsinogen-1, trypsinogen-2, trypsin-1-AAT, and trypsin-2-AAT were determined by time-resolved immunofluorometric assays. Logistic regression was used to estimate the ability of the serum analytes to discriminate between alcohol-induced and biliary AP. The validity of the tests was evaluated by ROC curve analysis. RESULTS: Patients with alcohol-induced AP had higher median values of trypsin-1-AAT (P = 0.065), trypsinogen-2 (P = 0.034), and trypsin-2-AAT (P <0.001) than those with biliary AP, who had higher values of amylase (P = 0.002), lipase (P = 0.012), and alanine aminotransferase (P = 0.036). The ratios of trypsin-2-AAT to trypsinogen-1, lipase, or amylase efficiently discriminated between biliary and alcohol-induced AP (areas under ROC curves, 0.92-0.96). CONCLUSIONS: Trypsinogen-2 and trypsin-2-AAT are markedly increased in AP of all etiologies, whereas trypsinogen-1 is increased preferentially in biliary AP. The trypsin-2-AAT/trypsinogen-1 ratio is a promising new marker for discrimination between biliary and alcohol-induced AP.

Acute Disease↗
PubMed2001

[Significance of trypsinogen gene mutations in the etiology of hereditary pancreatitis].

Hereditary, chronic pancreatitis is an autosomal dominant genetic disorder, frequently associated with two point mutations in the cationic trypsinogen gene. The mutations result in characteristic changes in the amino-acid sequence of trypsinogen: an arginine residue at position 117 is changed to histidine (Arg117-->His) or an asparagine residue at position 21 is replaced by isoleucine (Asn21-->Ile). Current opinion on the pathogenesis of hereditary pancreatitis suggests that the mutations lead to increased trypsin activity in the pancreatic tissue as a result of enhanced autoactivation of trypsinogen or decreased autocatalytic degradation (autolysis) of trypsin. To investigate the relationship between the altered properties of mutant trypsinogens and the pathomechanism of pancreatitis, wild-type and two mutant forms of recombinant human cationic trypsinogen were produced and autoactivation of trypsinogens and autolysis of trypsins were studied. The results indicate that trypsin stabilization (i.e. decreased autolysis) caused by the Arg117-->His mutation may contribute to the development of pancreatitis, however, the Asn21-->Ile mutation has no such effect. In contrast, enhanced autoactivation of mutant trypsinogens may contribute to the pathogenesis of both forms of hereditary pancreatitis. This notion is strongly supported by the clear correlation between the autoactivation rates of mutant trypsinogens and the severity of clinical symptoms.

Arginine↗
PubMed1996

Urine trypsinogen-2 as marker of acute pancreatitis.

We examined the clinical utility of urine trypsinogen-2 as a marker of acute pancreatitis (AP). Fifty-nine patients with AP, 42 with acute abdominal diseases of extrapancreatic origin, and 63 without evidence of acute abdominal disease were studied. Urine trypsinogen-2 was determined by a time-resolved immunofluorometric assay. As reference methods we used serum trypsinogen-2, urine amylase, and serum amylase. The diagnostic accuracy of the markers was evaluated by receiver-operating characteristic (ROC) analysis. At admission, urine trypsinogen-2 differentiated patients with AP from controls with high accuracy. The area under the ROC curve (AUC) was 0.978, which was equal to that of serum trypsinogen-2 (0.998) and serum amylase (0.969) and significantly larger than that of urine amylase. For differentiation between severe and mild AP, urine trypsinogen-2 (0.730) was equal to serum trypsinogen-2 (0.721), and clearly better than amylase in serum and urine. These results suggest that determination of urine trypsinogen-2 is a useful test to detect AP and to evaluate disease severity.

Acute Disease↗
PubMed2000

Down-regulation of trypsinogen-2 expression by chemically modified tetracyclines: association with reduced cancer cell migration.

Many types of human tumor express trypsinogen-2, which may be a significant factor in the activation of pro-MMPs and the invasiveness of tumors. Prevention of trypsinogen-2 expression in cancer cells might be of benefit in cancer therapy. We describe here chemicals capable of down-regulating the expression of trypsinogen-2. Doxycycline (DOXY) and chemically modified tetracyclines (CMTs), previously known as inhibitors of the matrix metalloproteinase (MMP)-dependent proteinase cascade, down-regulated the mRNA and protein expression of trypsinogen-2 by COLO-205 human colon adenocarcinoma cells at therapeutically attainable concentrations (0. 1 to 1.0 microM). DOXY specifically inhibited the activation of pro-MMP-9 and cell migration induced by enteropeptidase, a specific activator of trypsinogen. Pro-MMP-9 activation and cell migration were also inhibited by tumor-associated trypsin inhibitor (TATI), which is a highly specific inhibitor of trypsin. CMT-3 as well as CMT-5 also inhibited cell migration, but an effect on the enteropeptidase-enhanced activation of pro-MMP-9 was not observed. Our results indicate that CMTs, DOXY and TATI inhibit cancer cell migration by down-regulating trypsinogen-2 expression or activity. Inhibition of trypsinogen-2 expression may represent a mechanism contributing to the ability of CMTs to suppress the pericellular proteolytic activity of some tumors.

Antineoplastic Agents↗
PubMed2007

Detection of postoperative pancreatitis after pancreatic surgery by urine trypsinogen strip test.

BACKGROUND: The urine trypsinogen strip test has been used successfully in the diagnosis of pancreatitis of various aetiologies, but has not been studied in postoperative pancreatitis. The aim of this study was to evaluate this test for the diagnosis of postoperative pancreatitis after pancreatic resection. METHODS: Fifty patients undergoing pancreatic resection were included. The urine trypsinogen strip test was done daily during the first postoperative week, blood was analysed before and 4, 6 and 10 days after surgery, and amylase activity in the drainage fluid was measured on days 4 and 6. Patients underwent computed tomography (CT) before operation and on days 2 and 6 afterwards. RESULTS: Thirteen patients (26 per cent) developed CT-detected pancreatitis after operation. In 12 of these patients pancreatitis was detected on the second postoperative day. The urine trypsinogen test was positive in all 13 patients with postoperative pancreatitis, and was already positive on the first day after surgery in 12. The sensitivity, specificity, and positive and negative predictive values of the trypsinogen strip test in detection of postoperative pancreatitis were 100, 92, 81 and 100 per cent respectively. In receiver-operator characteristic analysis the area under the curve (AUC) was higher for the urine trypsinogen strip test (AUC 0.959) than for a serum amylase level more than two (AUC 0.731) or three times (AUC 0.654) above the upper normal range in the diagnosis of postoperative pancreatitis. Patients whose recovery was complicated by pancreatic fistula, detected by drain output measurements on day 6, more often had a positive urine trypsinogen test than patients without a fistula (11 of 12 versus five of 38; P < 0.001). CONCLUSION: This study suggests that the urine trypsinogen strip test might be a valuable method for diagnosis of pancreatitis after pancreatic surgery.

Adolescent↗
PubMed2001

Relationship between NF-kappaB and trypsinogen activation in rat pancreas after supramaximal caerulein stimulation.

Intra-acinar cell nuclear factor-kappaB (NF-kappaB) and trypsinogen activation are early events in secretagogue-induced acute pancreatitis. We have studied the relationship between NF-kappaB and trypsinogen activation in rat pancreas. CCK analogue caerulein induces early (within 15 min) parallel activation of both NF-kappaB and trypsinogen in pancreas in vivo as well as in pancreatic acini in vitro. However, NF-kappaB activation can be induced without trypsinogen activation by lipopolysaccharide in pancreas in vivo and by phorbol ester in pancreatic acini in vitro. Stimulation of acini with caerulein after 6 h of culture results in NF-kappaB but not trypsinogen activation. Protease inhibitors (AEBSF, TLCK, and E64d) inhibit both intracellular trypsin activity and NF-kappaB activation in caerulein stimulated acini. A chymotrypsin inhibitor (TPCK) inhibits NF-kappaB activation but not trypsin activity. The proteasome inhibitor MG-132 prevents caerulein-induced NF-kappaB activation but does not prevent trypsinogen activation. These findings indicate that although caerulein-induced NF-kappaB and trypsinogen activation are temporally closely related, they are independent events in pancreatic acinar cells. NF-kappaB activation per se is not required for the development of early acinar cell injury by supramaximal secretagogue stimulation.

Animals↗
PubMed1995

Ligand-induced changes in the conformational stability of bovine trypsinogen and their implications for the protein function.

Bovine trypsinogen was used as a model protein for studying changes in the conformational stability induced by pH or binding of the calcium ion. Spectrophotometrically monitored thermal unfolding of trypsinogen and beta-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to beta-trypsin, trypsinogen exhibits lower enthalpy of denaturation delta Hden, higher denaturational heat capacity change delta Cp,den, but very similar temperature of denaturation Tden. pH-dependence of the conformational stability of the ligand-free trypsinogen, measured also by GdnCl-induced unfolding, is bell shaped with the maximum free energy of unfolding delta Gden = 10.9 kcal/mole at pH 5.5 (4.5 pH units below its isoelectric point). At pH 8.3 the conformational stability of the zymogen drops to delta Gden = 3.2 kcal/mole, but increases by delta delta Gden = 6.1 kcal/mole in the presence of Ca2+. This significant stabilization of the zymogen by the calcium ion is also pH-dependent. To assess the effect of Ca2+ on the trypsinogen molecule, the spectrophotometric titrations and NOESY spectra were carried out. Based on the structural analysis, the long range effects between Ca2+-->Ile73-->Trp141 and the interdomain His40-Asp194 ion pair are proposed to be partially responsible for trypsinogen stabilization. Additionally, the steady-state parameters for hydrolysis of the oligopeptide amide substrate catalysed by free trypsinogen, its complexes with Ca2+ and the IleVal dipeptide and by beta-trypsin were measured. It appears that in the pH range 5.5 to 8.3 the stability and the catalytic activity/ligand binding properties are fully separated. Whereas the deprotonation of His57 accounts for the increase of kcat/km parameter, deprotonation of His40 is involved in the huge decrease of the conformational stability. Similarly, a large stabilization by the calcium ion is not accompanied by changes in enzymatic activity. Presented data are encouraging for an enzyme design directed toward improved stability.

Animals↗
PubMed1985

The identification of neotrypsinogens in samples of bovine trypsinogen.

Isoelectric focusing of commercial samples of bovine trypsinogen detected a component with a lower isoelectric pH than that of trypsinogen. The isoelectric pH was 8.75 compared to 9.3 for trypsinogen, and the amount of the component varied from 16 to 41% of the total protein. The protein (24,000 Da) was converted to fragments of 13,800 and 10,500 Da on reduction with dithioerythritol, showing that the component was a modified form of trypsinogen containing a cleaved peptide bond. The cleavage site was established from the study of four polypeptide fragments which were isolated from the fully reduced and S-carboxymethylated trypsinogen. The molecular weights, amino acid compositions, and amino-terminal sequences of these fragments identified a cleavage of Lys 131-Ser 132, namely from a Ser-neotrypsinogen, or at Arg 105-Val 106, from a Val-neotrypsinogen. Val-neotrypsinogen was the more abundant of the two and was approximately 71% of the total neotrypsinogen in the trypsinogen sample. Both neotrypsinogens were converted to active trypsin molecules in high yields, showing that the zymogens closely resembled the conformation of intact trypsinogen. Presumably, the neotrypsinogens were produced during the isolation of the zymogen when pancreatic tissue was partly autolyzed and active trypsin was present.

Amino Acid Sequence↗
PubMed1982

Purification and properties of multiple forms of mouse trypsinogen.

Three trypsinogens and one chymotrypsinogen were found in and purified from the pancreas of a mouse strain (CFO). The molecular weights of the trypsinogens and the chymotrypsinogen were all estimated as 25 000. The enzyme properties of the three trypsinogens were studied and they showed very low Km values (3.2-6.5 microM) for the substrates, Bz-Arg-OEt and Tos-Arg-OMe, and the same pH optimum profile between pH 8.0-10.0. However, the ratios of catalytic rate constants, kcat (s-1), with Bz-Arg-OEt as substrates compared to that with Tos-Arg-OMe were very different. The values of Try-III were similar with the two substrates, Try-I was slightly higher value with Tos-Arg-OMe than with Bz-Arg-OEt, and the values of Try-II were much higher with Tos-Arg-OMe than with Bz-Arg-OEt. Also, the trypsinogens and the chymotrypsinogen were purified from pancreas of Mol-A strain mice. When the enzyme properties of the three trypsinogens were examined, one form of trypsinogen (Try-I) was shown to have different properties in kcat (s-1) for the two substrates, compared to the trypsinogen of CFO mice.

Animals↗
PubMed2005

Cloning and expression of ostrich trypsinogen: an avian trypsin with a highly sensitive autolysis site.

One of ostrich (Struthio camelus) trypsinogen genes was cloned from pancreatic cDNA. Its amino acid sequence compared to known trypsin sequences from other species shows high identity and suggests that it is a member of the phylogenetically anionic trypsinogen I subfamily. After cytoplasmic over expression in Escherichia coli and renaturation, the activation properties of ostrich trypsinogen were studied and compared to those of human trypsinogen 1 (also called as human cationic trypsinogen). Ostrich trypsinogen undergoes bovine enterokinase activation and autoactivation much faster than human trypsinogen 1 and exhibits on a synthetic substrate a somewhat higher enzymatic activity than the latter one. The most interesting property of ostrich trypsin is its relatively fast autolysis that can be explained via a mechanism different from the common mechanism for rat and human 1 trypsins. The latter proteases have a site, Arg117-Val118, where the autolysis starts and then goes on in a zipper-like fashion. This is absent from ostrich trypsin. Instead it has a couple of cleavage sites within regions 67-98, including two unusual ones, Arg76-Glu77 and Arg83-Ser84. These appear to be hydrolysed fast in a non-consecutive manner. Such an autolysis mechanism could not be inhibited by a single-site mutation which in humans is proposed to lead to pancreatitis.

Amino Acid Sequence↗
PubMed1996

Serum samples from pancreatectomized patients contain trypsinogen immunoreactivity.

The concentrations of trypsinogen-1 and -2 in serum samples from patients who have undergone pancreatectomy were measured by highly sensitive and specific time-resolved immunofluorometric assays. The isoenzyme pattern was determined by ion-exchange chromatography and determination of immunoreactivity in the fractions. All samples contained trypsinogen-2, the mean level being one fifth of that in healthy controls. Trypsinogen-1 was detected in one of nine samples. In addition to the main trypsinogen isoenzymes, we observed in normal serum two trypsinogen isoenzymes previously found in mucinous ovarian cyst fluid. Our results suggest that trypsinogen is not exclusively expressed by the pancreas and certain tumors but that it also may be produced by normal extrapancreatic tissues. This should be considered when an assay of trypsinogen in serum is used for clinical purposes.

Adenocarcinoma↗
PubMed1985

Elevated serum immunoreactive pancreatic cationic trypsinogen in acute malnutrition: evidence of pancreatic damage.

We used a sensitive probe of pancreatic dysfunction, serum immunoreactive cationic trypsinogen, to study 50 infants and children with varying degrees of malnutrition. Patients were classified into subgroups according to the severity of malnutrition. Mean serum trypsinogen concentration was significantly elevated in 25 patients with "severe" malnutrition (77.4 +/- 42.0 ng/ml, P less than 0.001) and in 23 with "moderate" malnutrition (55.2 +/- 16.1 ng/ml, P less than 0.02) compared with the mean value (32.5 +/- 10.4 ng/ml) for well-nourished controls. The level of circulating trypsinogen tended to rise with increasing severity of malnutrition. There was no relationship between serum trypsinogen and other variables such as age, specific diagnosis, or mode of feeling. Elevated serum trypsinogen levels could not be attributed to renal disease or cystic fibrosis. In patients who showed an improvement in nutritional status, serum trypsinogen tended to revert toward normal. Elevated serum trypsinogen values in acutely malnourished infants and children may result from pancreatic acinar cell damage or regurgitation of enzymes from obstructed pancreatic ducts.

Acute Disease↗
PubMed1978

Two human trypsinogens. Purification, molecular properties, and N-terminal sequences.

The two human trypsinogens have been isolated from human pancreatic juice in a sufficient amount to study molecular and structural properties. The purification procedure included filtration on Sephadex G-100 followed by ion-exchange chromatography on DEAE-cellulose. The two trypsinogens represent 19% of total proteins of pancreatic juice. Trypsinogen 1, the major form, is present in a quantity twice that of trypsinogen 2, which is the most anionic protein in human pancreatic juice. The two proteins have partial immunological identity, close molecular weights (23 438 and 25 006 for trypsinogens 1 and 2, respectively) and similar amino acid compositions. The N-terminal sequences are the same for the first 9 residues: Ala-Pro-Phe-Asp4-Lys-Ile. The two proteins differ in the activation peptides released during the transformation to trypsins. Trypsinogen 2 liberates one octapeptide Ala-Pro-Phe-Asp4-Lys while trypsinogen 1 liberates two peptides, the same octapeptide and the pentapeptide (Asp)4-Lys.

Amino Acid Sequence↗
PubMed1994

Pancreatic trypsinogen and cathepsin B in human pancreatic carcinomas and associated metastatic lesions.

Expression of pancreatic trypsinogen and cathepsin B in 23 surgically resected pancreatic ductal adenocarcinomas was evaluated immunohistochemically, using a monoclonal antibody against human pancreatic trypsinogen and a polyclonal antibody against human cathepsin B. Fifteen of 20 invasive tubular adenocarcinomas (75%) expressed pancreatic trypsinogen in a coarse granular pattern located in the supranuclear cytoplasm of the carcinoma cells. In addition, metastatic lesions, including those in peripancreatic lymph nodes and neural plexuses, expressed pancreatic trypsinogen. In contrast, three intraductal (non-invasive) papillary adenocarcinomas did not express pancreatic trypsinogen. Cathepsin B expression was recognised in 14 of 20 invasive tubular adenocarcinomas (70%) in a fine granular pattern located diffusely in the cytoplasm of the carcinoma cells, while none of the three intraductal papillary adenocarcinomas had detectable cathepsin B. These findings suggest that pancreatic invasive ductal adenocarcinomas express pancreatic trypsinogen and cathepsin B immunoreactive peptides, raising the possibility that pancreatic trypsinogen and cathepsin B may act independently of each other in the process of carcinoma invasion and metastasis, like other different classes of proteases involved in the proteolytic modification of the matrix barrier.

Adult↗
PubMed1997

Urinary trypsinogen-2 test strip in detecting ERCP-induced pancreatitis.

BACKGROUND AND STUDY AIMS: We have evaluated a new urinary trypsinogen-2 test strip, based on the principle of immunochromatography, in the diagnosis of acute pancreatitis induced by endoscopic retrograde cholangiopancreatography (ERCP). PATIENTS AND METHODS: One hundred six consecutive patients undergoing ERCP (with opacification of the pancreatic duct) at the Helsinki University Central Hospital were included in the study. Patients were tested with a urinary trypsinogen-2 test strip six hours after ERCP. Quantitative trypsinogen-2 as well as serum and urine amylase values were measured before the procedure and six hours after it. RESULTS: In patients developing pancreatitis after ERCP, the median urinary trypsinogen-2 concentration six hours after the endoscopic procedure was 1780 micrograms/l (range 29-10,700 micrograms/l), and in patients without pancreatitis the median concentration was 3.6 micrograms/l (range 0.1-3390 micrograms/l; P < 0.0001). The sensitivity and specificity figures for the urinary trypsinogen-2 test strip results in diagnosing post-ERCP pancreatitis were comparable (81% and 97%, respectively) to those for serum amylase (91% and 96%) and urine amylase measurements (81% and 95%). The test strip showed a good correlation (kappa = 0.75) with the quantitative trypsinogen-2 assay. CONCLUSIONS: The increase in urinary trypsinogen-2 concentration after ERCP reflects pancreatic injury, and can be detected by the test strip. Patients should be tested before the ERCP procedure as well, since elevated baseline values occur. The test is reliable and easy to perform even on an outpatient basis. However, its clinical usefulness requires evaluation in further trials.

Acute Disease↗
PubMed1997

Rapid measurement of urinary trypsinogen-2 as a screening test for acute pancreatitis.

BACKGROUND: Acute pancreatitis can be difficult to diagnose. We developed a rapid dipstick screening test for pancreatitis, based on the immunochromatographic measurement of urinary trypsinogen-2. METHODS: We prospectively compared the urinary trypsinogen-2 dipstick test with a quantitative urinary trypsinogen-2 assay, a urinary dipstick test for amylase, and serum and urinary amylase assays in 500 consecutive patients with acute abdominal pain at two emergency departments. Acute pancreatitis was diagnosed according to standardized criteria. RESULTS: The urinary trypsinogen-2 dipstick test was positive in 50 of the 53 patients with acute pancreatitis (sensitivity, 94 percent), including all 7 with severe pancreatitis. Two patients with urinary trypsinogen-2 concentrations below the sensitivity threshold of the test (50 ng per milliliter) and one with a very high concentration had false negative results. The test was also positive in 21 of the 447 patients without pancreatitis (specificity, 95 percent), including 7 with abdominal cancers, 3 with cholangitis, and 2 with chronic pancreatitis. The sensitivity and specificity of the dipstick test were similar to those of the quantitative urinary trypsinogen-2 assay and higher than those of the urinary amylase dipstick test. The serum amylase assay had a sensitivity of 85 percent (with a cutoff value of 300 U per liter for the upper reference limit) and a specificity of 91 percent. The sensitivity and specificity of the urinary amylase assay (cutoff value, 2000 U per liter) were 83 and 88 percent, respectively. CONCLUSIONS: In patients with acute abdominal pain seen in the emergency department, a negative dipstick test for urinary trypsinogen-2 rules out acute pancreatitis with a high degree of probability. A positive test usually identifies patients in need of further evaluation.

Acute Disease↗
PubMed1999

Hereditary pancreatitis-associated mutation asn(21) --> ile stabilizes rat trypsinogen in vitro.

Mutations Arg(117) --> His and Asn(21) --> Ile in human trypsinogen-I have been recently associated with hereditary pancreatitis (HP). The Arg(117) --> His substitution is believed to cause pancreatitis by stabilizing trypsin against autolytic degradation, while the mechanism of action of Asn(21) --> Ile has been unknown. In an effort to understand the effect(s) of this mutation, Thr(21) in the highly homologous rat trypsinogen-II was replaced with Asn or Ile, and the recombinant zymogens and their active trypsin forms were studied. Kinetic parameters of all three trypsins were comparable, and the active enzymes suffered autolysis at similar rates, indicating that neither catalytic properties nor proteolytic stability of trypsin are influenced by mutations at position 21. When incubated at pH 8.0, 37 degrees C, pure zymogens underwent autoactivation with concomitant trypsinolytic degradation in a Ca(2+)-dependent fashion. Thus, in the presence of 5 mM Ca(2+), autoactivation and digestion of the zymogens after Arg(117) and Lys(188) were observed, while in the presence of 1 mM EDTA autoactivation and cleavage at Lys(188) were reduced, and zymogenolysis at the Arg(117) site was enhanced. Overall rates of zymogen degradation in [Asn(21)]- and [Ile(21)]trypsinogens were higher in Ca(2+) than in EDTA, while [Thr(21)]trypsinogen demonstrated inverse characteristics. Remarkably, both in the presence and absence of Ca(2+), [Ile(21)]trypsinogen exhibited significantly higher stability against autoactivation and proteolysis than zymogens with Asn(21) or Thr(21). The observations suggest that autocatalytic trypsinogen degradation may be an important defense mechanism against excessive trypsin generation in the pancreas, and trypsinogen stabilization by the Asn(21) --> Ile mutation plays a role in the pathogenesis of HP.

Animals↗