Anionic Trypsinogen Research Articles

Differential regulation of trypsinogen mRNA translation: full-length mRNA sequences encoding two oppositely charged trypsinogen isoenzymes in the dog pancreas.

In the absence of changes in functional mRNA levels, stimulation of the pancreas with caerulein, a peptide analog of cholecystokinin, has been previously shown to increase the synthesis of anionic but not cationic trypsinogen. To look for structure-function correlations, a high-yield, full-length cDNA library has been constructed from canine pancreatic poly(A)+ mRNA. Full-length clones coding for the two major trypsinogen isoenzyme forms have been identified by colony hybridization and verified by in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and an optimal redox potential. Disulfide-bonded translation products were separated and identified by two-dimensional isoelectric focusing-sodium dodecyl sulfate-gel electrophoresis. Nucleotide sequence analysis allowed us to deduce the amino acid sequences for the anionic and cationic forms of canine trypsinogen, which contain 232 and 231 residues, respectively (77% amino acid identity), and the 15-residue amino terminal signal sequences (53% amino acid identity) associated with the two presecretory forms. Measurements of relative and absolute mRNA levels, when related to relative protein synthesis values, indicated that the translational efficiency of anionic trypsinogen mRNA exceeded that of cationic trypsinogen mRNA by 1.5- to 2.9-fold under basal conditions. Analysis of the 5' noncoding regions of trypsinogen mRNAs revealed a striking conservation of sequence (10 of 12 bases) between dog and rat anionic trypsinogen forms. This contrasted markedly with the divergence of the 5' noncoding regions observed between dog anionic and cationic trypsinogen mRNAs.

Differential regulation of trypsinogen mRNA translation: full-length mRNA sequences encoding two oppositely charged trypsinogen isoenzymes in the dog pancreas.

In the absence of changes in functional mRNA levels, stimulation of the pancreas with caerulein, a peptide analog of cholecystokinin, has been previously shown to increase the synthesis of anionic but not cationic trypsinogen. To look for structure-function correlations, a high-yield, full-length cDNA library has been constructed from canine pancreatic poly(A)+ mRNA. Full-length clones coding for the two major trypsinogen isoenzyme forms have been identified by colony hybridization and verified by in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and an optimal redox potential. Disulfide-bonded translation products were separated and identified by two-dimensional isoelectric focusing-sodium dodecyl sulfate-gel electrophoresis. Nucleotide sequence analysis allowed us to deduce the amino acid sequences for the anionic and cationic forms of canine trypsinogen, which contain 232 and 231 residues, respectively (77% amino acid identity), and the 15-residue amino terminal signal sequences (53% amino acid identity) associated with the two presecretory forms. Measurements of relative and absolute mRNA levels, when related to relative protein synthesis values, indicated that the translational efficiency of anionic trypsinogen mRNA exceeded that of cationic trypsinogen mRNA by 1.5- to 2.9-fold under basal conditions. Analysis of the 5' noncoding regions of trypsinogen mRNAs revealed a striking conservation of sequence (10 of 12 bases) between dog and rat anionic trypsinogen forms. This contrasted markedly with the divergence of the 5' noncoding regions observed between dog anionic and cationic trypsinogen mRNAs.

The exocrine protein trypsinogen is targeted into the secretory granules of an endocrine cell line: studies by gene transfer.

The exocrine protein rat anionic trypsinogen has been expressed and is secreted from the murine anterior pituitary tumor cell line AtT-20. We examined which secretory pathway trypsinogen takes to the surface of this endocrine-derived cell line. The "constitutive" pathway externalizes proteins rapidly and in the absence of an external stimulus. In the alternate, "regulated" pathway, proteins are stored in secretory granules until the cells are stimulated to secrete with 8-Br-cAMP. On the basis of indirect immunofluorescence localization, stimulation of release, and subcellular fractionation, we find that trypsinogen is targeted into the regulated secretory pathway in AtT-20 cells. In contrast, laminin, an endogenous secretory glycoprotein, is shown to be secreted constitutively. Thus it appears that the transport apparatus for the regulated secretory pathway in endocrine cells can recognize not only endocrine prohormones, but also the exocrine protein trypsinogen, which suggests that a similar sorting mechanism is used by endocrine and exocrine cells.

Effects of food intake and cholecystokinin on plasma trypsinogen levels in dogs.

Concentrations of plasma immunoreactive anionic and cationic trypsin(ogen) were monitored in unanesthetized dogs to investigate diurnal variation, response to food intake, and effects of cholecystokinin octapeptide (CCK-8) plus secretin administration. Identical meals were consumed at the beginning and end of a 24-h period. Plasma levels of both trypsin(ogen)s increased significantly within 30 min of feeding. Second-meal responses were much larger than those following the first meal. Plasma cationic trypsin(ogen) was also elevated during the normal feeding period when food was withheld and dogs in adjacent cages were fed. Small, irregular variations in plasma trypsin(ogen)s occurred during the interdigestive period when blood was sampled every 2 h. CCK-8 plus secretin, in doses shown previously to stimulate exocrine secretion of enzymes submaximally, increased plasma levels of both trypsin(ogen)s dose dependently. Most (greater than 80%) of the immunoreactive cationic trypsin(ogen) in plasma before and after feeding and following CCK-8 was in proenzyme form. We conclude that plasma levels of pancreatic trypsinogens are increased by food intake and low doses of CCK-8. Furthermore, cephalic mechanisms appear to play an important role in mediating the response to food intake.

Mesotrypsin: A New Inhibitor-Resistant Protease From, a Zymogen in Human Pancreatic Tissue and Fluid

We have isolated and identified a new zymogen in human pancreatic tissue and fluid. It is secreted as a minor component of pancreatic juice and resembles the two known trypsinogen variants in many properties. Its electrophoretic mobility and isoelectric pH lie between those of the cationic and anionic trypsinogen variants, and we propose the name "mesotrypsinogen" for the new enzyme precursor. It is activated by enteropeptidase or trypsin, and the free enzyme possesses a substrate specificity similar to that of the trypsins. Its pH optimum is at 8.2, and it appears to require Ca2+ for full enzymatic activity. The molecular weight of the new enzyme is approximately 25,000, similar to that of the known trypsin variants. Its stability resembles that of anionic trypsin extending over a pH range of 4-8.5. Activity is lost gradually at pH 2. The enzyme is inactivated rapidly by diisopropylfluorophosphate, but in contrast to the trypsins, it reacts only slowly with tosyllysine chloromethylketone. Immunologically, it is different from the cationic trypsin variant with which it does not cross-react. The most remarkable property of mesotrypsin is its almost total resistance to biological trypsin inhibitors, such as pancreatic trypsin inhibitor, soybean, lima bean, ovomucoid inhibitor, alpha 1-antitrypsin, etc. It is capable of activating trypsinogen in the presence of excess pancreatic trypsin inhibitor and thus inducing activation of other pancreatic zymogens, but it also possesses the ability to degrade trypsinogen rapidly to inert products. The physiological or pathophysiological role of this unique enzyme remains to be explored.

Dog pancreatic microsomal-membrane polypeptides analysed by two-dimensional gel electrophoresis.

The two-dimensional polyacrylamide-gel electrophoresis technique of O'Farrell [(1975) J. Biol. Chem 250, 4007-4021] was applied to resolve and analyse the polypeptide composition of dog pancreatic rough microsomal membranes, which were shown to be active in co-translational processing of preprolactin synthesized from pituitary mRNA in a translation system in vitro. About 100 polypeptides are resolved. Treatment of rough microsomal membranes with EDTA and high KCl concentration yielded membranes stripped of their ribosomes with retention of activity for translocation and processing. Stripped microsomal membranes showed a selective concentration of approximately 25 polypeptides in the membranes when analysed by two-dimensional polyacrylamide-gel electrophoresis. The two-dimensional electrophoretic profile was catalogued into polypeptides that are glycoproteins, those that contain free thiol groups disposed at the cytosolic surface of microsomal vesicles and those that are of secretory origin but have been entrapped in the microsomal preparation. Several secretory components, including amylase, procarboxypeptidases, lipase and anionic trypsinogen, were tentatively identified among the microsomal polypeptides. The rough and stripped microsomal membranes from dog pancreas show a characteristic set of seven major acidic polypeptides, which are also identifiable in microsomal-membrane preparations isolated from dog liver and rat liver. One of these polypeptides was identified as protein disulphide-isomerase (EC 5.3.4.1).

A simple procedure for the purification of the zymogens and active forms of canine anionic trypsin, cationic trypsin and chymotrypsin

In order to prepare canine trypsins (EC 3.4.21.4) for use in radioimmunoassay, a convenient method for their purification was required. Amnity chromatography has been used with considerable success in the purification of serine proteinases. Naturally occurring inhibitors such as chicken ovomucoid, bovine pancreatic trypsin inhibitor and lima-bean trypsin inhibitor have facilitated the resolution of bovine uand &trypsins (Robinson et al., 1971), human anionic and cationic trypsins (Malvano et al., 1979), and human cationic trypsinogen (Brodrick et al., 1978). Additionally, synthetic ligands such as proflavin (Brantner et al., 1976) and 4-aminobenzamidine (Schmer, 1972) have been shown to bind serine proteinases, and thus several potential options exist for the purification of canine trypsins. The present communication reports that 4-aminobenzamidine, covalently linked to agarose beads via a 6amino-n-hexanoic acid spacer (Schmer, 1972), is a suitable aflinity matrix for this purpose, and in addition may be used to purify the trypsinogens. The matrix also retards the passage of chymotrypsin (EC 3.4.21.1) and chymotrypsinogen. Pancreas (1OOg) stored frozen at -2OOC was homogenized in 0.125 M-H,SO,, and the supernatant was fractionated between 0 . 8 ~ and 3.0hi-(NH4),S04 (Travis & Roberts, 1969). The precipitate was dissolved in water, dialysed exhaustively against ImM-HCI and freeze-dried to yield approx. 1.5g of crude extract. Gel filtration on a 90cm x 3.8cm2 column of Sephadex G-75 in 0.2 M-NaCI/O.OS M-CaCl, medium pH 2.6, yielded two major elution peaks. The second peak contained the zymogens, and these fractions were dialysed and freeze-dried. Pancreatic secretory trypsin inhibitor (Fritz, 1967) was eluted in a minor peak immediately after the zymogens. The fractions containing the pancreatic trypsin inhibitor were pooled and adjusted to pH9.0 by addition of NaOH to give a crude inhibitor solution. The partially purified zymogens were further resolved by dissolving the freeze-dried residue in solution containing the pancreatic trypsin inhibitor and applying this extract in SOmM-Tris buffer, pH9.75, containing 2m~-CaC1, to the aflinity matrix in a 12cm x 0.8cm2 column. Chymotrypsinogen was retarded, but was eluted rapidly with buffer containing 0.1 M-NaCl. Cationic trypsinogen was desorbed with buffer containing 0.2 M-NaCl. Anionic trypsinogen was eluted slowly by high-ionic-strength solutions, but was desorbed readily with 50mM-sodium acetate buffer, pH 3.75, containing 2mM-CaC1,. To resolve active enzymes the partially purified zymogens obtained by gel filtration were allowed to autoactivate for 12 h at 4°C in 0.1 M-TI-~s buffer, pH8, containing 4m~-CaCl , in the absence of the pancreatic trypsin inhibitor. This extract was applied to the affinity matrix in SOmM-Tris buffer, pH8.0, containing 21AM-CaC1,. Chymotrypsin was desorbed with the same buffer containing O.SwNaC1. Cationic trypsin was eluted with 50mM-sodium acetate buffer, pH4.25, containing 2 mMCaCl,, and anionic trypsin was eluted with 5OmM-sodium acetate buffer, pH 3.25, containing 2mwCaC1,. Typical purification schemes for the zymogen and active forms of anionic and cationic trypsin are shown in Table 1. Trypsin and chymotrypsin were assayed with benzoylarginine ethyl ester and acetyltyrosine ethyl ester respectively (Schwert & Table 1. Purijication of the zymogens and active forms of canine anionic and cationic trypsins

Plasma Immunoreactive Anionic Pancreatic Trypsin in Cystic Fibrosis

SummaryPrevious data from this laboratory have shown good correlation between plasma cationic trypsin(ogen) and levels of pancreatic function in older cystic fibrosis (CF) patients with and without malabsorption. In the present study a radioimmunoassay for human anionic trypsin(ogen) has been employed in the assessment of pancreatic function in older patients with CF. Immunoreactive anionic trypsin(ogen) levels correlated poorly with pancreatic function due to an apparent elevation of this enzyme in many older CF patients with malabsorption (CF + M). When plasma from three older CF + M patients is examined for the molecular size of the apparent immunoreactive material detected, no free anionic trypsinogen is observed. Instead, a broad peak of apparent immunoreactive material appears in the gammaglobulin region. However, only free anionic trypsinogen could be detected in plasma from two CF patients without malabsorption (CF ‐ M), who had not received pancreatic enzyme supplements. It appears possible that a human plasma immunoglobulin G (IgG) to porcine pancreatic enzyme in the CF + M patients might interfere in the assay by binding TLCK‐anionic trypsin tracer. It is unclear why such an effect does not appear to occur in the cationic trypsin(ogen) assay. The results of the current study suggest that, for assessment of pancreatic insufficiency in CF, the radioimmunoassay for cationic trypsin(ogen) is more useful than the presently available radioimmunoassay for the anionic form.

Plasma pancreatic trypsinogens in chronic renal failure and after nephrectomy.

The kidney has previously been shown to be a major site for the plasma clearance of pancreatic trypsinogens in the rat. This study investigated plasma concentrations of anionic and cationic trypsinogen in chronic renal failure and anephric patients. Plasma concentrations were significantly elevated in both groups of patients. Hemodialysis did not change their plasma levels. The plasma levels of anionic and cationic trypsinogens were highly correlated in patients and normal subjects; however, the relative concentrations of anionic trypsinogen were significantly higher in renal failure patients. This suggests that in patients with renal failure the secondary clearance mechanisms for these plasma proteins more efficiently clear cationic molecules. In normal dogs, intravenous infusion of synthetic octapeptide of cholecystokinin (CCK-8) resulted in small transitory increases in plasma trypsinogen levels. After nephrectomy, basal levels of anionic and cationic trypsinogen were elevated, and intravenous infusion of CCK-8 resulted in prolonged, high levels of plasma trypsinogens.

Plasma immunoreactive anionic pancreatic trypsin in cystic fibrosis.

Previous data from this laboratory have shown good correlation between plasma cationic trypsin(ogen) and levels of pancreatic function in older cystic fibrosis (CF) patients with and without malabsorption. In the present study a radioimmunoassay for human anionic trypsin(ogen) has been employed in the assessment of pancreatic function in older patients with CF. Immunoreactive anionic trypsin(ogen) levels correlated poorly with pancreatic function due to an apparent elevation of this enzyme in many older CF patients with malabsorption (CF + M). When plasma from three older CF + M patients is examined for the molecular size of the apparent immunoreactive material detected, no free anionic trypsinogen is observed. Instead, a broad peak of apparent immunoreactive material appears in the gammaglobulin region. However, only free anionic trypsinogen could be detected in plasma from two CF patients without malabsorption (CF - M), who had not received pancreatic enzyme supplements. It appears possible that a human plasma immunoglobulin G (IgG) to porcine pancreatic enzyme in the CF + M patients might interfere in the assay by binding TLCK--anionic trypsin tracer. It is unclear why such an effect does not appear to occur in the cationic trypsin(ogen) assay. The results of the current study suggest that, for assessment of pancreatic insufficiency in CF, the radioimmunoassay for cationic trypsin(ogen) is more useful than the presently available radioimmunoassay for the anionic form.

The fate of intravenously injected trypsinogens in dogs.

After intravenous injection of autologous purified and radiolabeled anionic and cationic trypsinogens in the dog, there is a multiexponential elimination from serum of the trypsinogens without activation to active trypsins. The results are in agreement with an elimination of the trypsinogens iva glomerular filtration, a variable tubular uptake of anionic trypsinogen, and a complete tubular uptake of cationic trypsinogen. Fractional catabolic rates for anionic trypsinogen were 0.35-0.92 h-1 and for cationic trypsinogen 0.38-1.05 h-1. The total amount of anionic trypsinogen turned over in the circulation is estimated at approximately 0.55 mg every 24 h, and the corresponding figure for cationic trypsinogen is 0.35 mg/24 h.

Clearance of circulating anionic and cationic pancreatic trypsinogens in the rat.

The kinetics and mechanism of clearance of pancreatic cationic and anionic trypsinogens from the circulation have been investigated in a rat model. 125I-labeled rat cationic trypsinogen is cleared from the bloodstream with a half-life of approximately 4 min. In contrast, 125I-labeled rat anionic trypsinogen has a half-life in the circulation of approximately 55-60 min. The major site of clearance for both enzymes is the kidney. Neither zymogen binds to plasma proteins to a significant extent over a period of three half-lives. The relative rates of clearance of these zymogens from the circulation appears to correlate with their respective isoelectric points.

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, α-macroglobulin and α 1-antitrypsin. α-Macroglobulin cannot inhibit the activation of the trypsinogens by trypsin but this reaction is completely inhibited by α 1-antitrypsin. The results are discussed in relation to the pathogenesis of acute pancreatitis.

Trypsinogen variants in pancreatic juice of healthy volunteers, chronic alcoholics, and patients with pancreatitis and cancer of the pancreas.

Polyacrylamide gel electrophoresis of pure pancreatic juice from 14 healthy normal subjects, 11 chronic alcoholics without detectable pancreatic disease, 15 patients with pancreatitis, and two with cancer of the pancreas consistently demonstrated the presence of two variants of trypsinogen with different electrophoretic mobilities. In healthy normal subjects the proportion of cationic to anionic trypsinogen was invariably greater than 1 and averaged about 2. In chronic alcoholics, patients with pancreatitis or cancer of the pancreas, this ratio, with a single exception, was below one and averaged about 0.45. The extraordinary consistency of these findings suggests that the quantitative relationship between cationic and anionic trypsinogen in human pancreatic juice may be a very sensitive indicator of incipient or existing pancreatic pathology. The most acceptable explanation for the reversal of the normal zymogen ratio in pancreatic disease is a selective increase in the synthesis of the anionic variant relative to that of the cationic species. Total trypsinogen concentrations differed widely from one another in the three patient groups, but the ratio of cationic to anionic trypsinogen exhibited little change and remained below 1. Our results also demonstrate for the first time a specific effect of chronic alcohol abuse on the secretory profile of a pancreatic enzyme in human subjects. A newly discovered minor, trypsinogen-like component of human pancreatic juice was found to be significantly increased in pancreatic juice of chronic alcoholics, decreased in pancreatic secretions of patients with pancreatitis, and barely detectable in those of two patients with cancer of the pancreas.

Purification and N-terminal amino acid sequence determination of anionic and cationic canine trypsinogens.

Anionic and cationic canine trypsinogens were purified from pancreatic juice by affinity chromatography with Trasylol coupled to Sepharose 4B followed by ion exchange chromatography with SP-Sephadex C-50. Automatic N-terminal amino acid sequence determination showed the following structures for the activation peptides: Thr-Pro-Thr-Asp-Asp-Asp-Asp-Lys for anionic trypsinogen, and Phe-Pro-Ile-Asp-Asp-Asp-Asp-Lys for cationic trypsinogen.

Demonstration of human pancreatic anionic trypsinogen in normal serum by radioimmunoassay

A specific radioimmunoassay for human pancreatic anionic trypsin has been developed. The trypsin employed as radioiodinated tracer in the assay was inactivated with tosyl- L-lysine chloromethyl ketone in order to prevent binding of the tracer to the serum inhibitors α 1-antitrypsin and α 2-macroglobulin. A normal serum level of immunoreactive anionic trypsin of 5.45 ng/ml was determined. The results of experiments in which serum was fractionated by Sephadex G-200 gel filtration suggest that essentially all of the immunoreactive material in normal human serum is trypsinogen. This finding implies that a small fraction of the zymogens synthesized in the pancreas are released directly into the circulation.

On bovine and porcine anionic trypsinogens

The proportions of the anionic trypsinogens in cattle and pig pancreas do not exceed 10% of those of the well known cationic forms. Their amino acid compositions differ markedly, suggesting a relatively low degree of homology between both classes. The anionic trypsinogen from pig pancreas yielded after activation by entero-kinase a single peptide with 8 residues which was identical to that detached during the activation of the cationic zymogen in the same species. By contrast, activated cattle anionic trypsinogen yielded, not only a major octapeptide (90%) but also an hexapeptide (10%). The kinetic parameters of the enterokinase-catalyzed activation of the anionic trypsinogens appear to be distinctly more favorable than for their cationic analogs. This may compensate their lower proportions in the duodenum.

Anionic and cationic dog trypsin. Isolation and partial characterization

One anionic and one cationic dog trypsin have been identified in dog pancreatic juice and isolated. The dog pancreatic juice contained 1.4 mg/ml of anionic trypsinogen and 1.0 mg/ml of cationic trypsinogen, respectively. The conversion of the trypsinogens to active enzymes is autocatalytic with the formation of active enzymes with higher isoelectric points than the zymogens. Although the amino acid compositions of the two trypsins were similar to those of other mammalian trypsins, a significant difference in content of acidic and basic amino acids, respectively, was found between the two dog trypsins. A high extent of cross-inhibition of the dog trypsins and of bovine trypsin by antibodies to each of the dog trypsins indicated immunological cross-reaction between the various trypsins despite lack of demonstrable cross-precipitation in gels. The calculated s 0 20, w value for both dog trypsins was 3.3 S. The K m value of anionic trypsin for benzoyl- d,l-arginine -p- nitroanilide · HCl was found to be similar to that of cationic trypsin and to that of bovine cationic trypsin. Soybean trypsin inhibitor, chicken ovomucoid, porcine and bovine secretory inhibitors are potent inhibitors of anionic and cationic dog trypsin.

Composition of human pancreatic juice. Immunoenzymological studies

The enzyme composition of human pancreatic juice has been studied by immunological techniques, using non‐activated and activated juice. Antisera were prepared with activated and non‐activated pancreatic juice. Same samples of antisera were adsorbed with human serum. Immunoelectrophoresis was carried out at pH 8.6 and disc immunoelectrophoresis at pH 8.9 and 5.0. Gel‐diffusion media contained benzamidine, to prevent zymogen activation. Precipitation lines were characterized by activating the zymogens on the plates with trypsin, and identifying the enzymes produced using specific synthetic substrates. Immunoelectrophoresis showed 15–20 antigenic constituents. Although disc immunoelectrophoresis gave slightly different results, those were consistent with the results of immunoelectrophoresis. The following active enzymes were identified and localized: α‐amylase, lipase and carboxylester hydrolase, which is a lipolytic enzyme structurally and catalytically different from lipase. Several zymogens were identified: two immunologically distinct trypsinogens, one procarboxypeptidase B, two forms of procarboxypeptidase A, which are immunologically identical, and two proelastases with some common antigenic determinants. After activation, both proelastases showed a weak but different degree of chymotryptic activity. Chymotrypsinogen was localized in two major anionic lines, and one, minor, cationic line. The possibility that one anionic immune precipitate represents a polymer is discussed. No chymotryptic activity was associated with procarboxypeptidase A. Immunoelectrophoresis of activated pancreatic juice demonstrates electrophoretic and antigenic changes. Activation of one anionic trypsinogen gave rise to cationic trypsin, whereas after activation chymotrypsinogens and procarboxypeptidases A showed no important change.

Identification of an anionic trypsinogen in bovine pancreas

A strongly anionic trypsinogen has been identified in bovine pancreas with the aid of enterokinase as the activating agent. The new zymogen has been substantially purified by two chromatographies on DEAE-cellulose.