Presence of cathepsin B in the human pancreatic secretory pathway and its role in trypsinogen activation during hereditary pancreatitis

The lysosomal cysteine protease cathepsin B is thought to play a central role in intrapancreatic trypsinogen activation and the onset of experimental pancreatitis. Recent in vitro studies have suggested that this mechanism might be of pathophysiological relevance in hereditary pancreatitis, a human inborn disorder associated with mutations in the cationic trypsinogen gene. In the present study evidence is presented that cathepsin B is abundantly present in the secretory compartment of the human exocrine pancreas, as judged by immunogold electron microscopy. Moreover, pro-cathepsin B and mature cathepsin B are both secreted together with trypsinogen and active trypsin into the pancreatic juice of patients with sporadic pancreatitis or hereditary pancreatitis. Finally, cathepsin B- catalyzed activation of recombinant human cationic trypsinogen with hereditary pancreatitis-associated mutations N29I, N29T, or R122H were characterized. In contrast to a previous report, cathepsin B-mediated activation of wild type and all three mutant trypsinogen forms was essentially identical under a wide range of experimental conditions. These observations confirm the presence of active cathepsin B in the human pancreatic secretory pathway and are consistent with the notion that cathepsin B-mediated trypsinogen activation might play a pathogenic role in human pancreatitis. On the other hand, the results clearly demonstrate that hereditary pancreatitis-associated mutations do not lead to increased or decreased trypsinogen activation by cathepsin B. Therefore, mutation-dependent alterations in cathepsin B-induced trypsinogen activation are not the cause of hereditary pancreatitis.     

Sepharose-bound trypsin and activated sepharose-bound trypsinogen. Studies with small and large substrates

Several enzymic and physical properties of Sepharose-bound trypsin and activated Sepharose-bound trypsinogen have been compared to those of the soluble enzyme. Sepharose-bound trypsinogen could be activated to the same extent as soluble trypsinogen; the release of the activation peptide and formation of the active site occurred as expected in the presence of catalytic amounts of trypsin. With synthetic substrates, the relative activity and pH dependence of both immobilized trypsin preparations were essentially identical and nearly the same as the soluble enzyme. Sepharose-trypsin also formed an inactive complex with soybean trypsin inhibitor, with 85% of the active sites participating. In contrast, the activity of Sepharose-trypsin with chymotrypsinogen and with trypsinogen as substrates was only 40% that of soluble trypsin. There is evidence for some catalytic heterogeneity of active sites of bound trypsin; probably those sites buried within the gel have a limited catalytic efficiency with macromolecular substrates. The immobilized enzyme is more stable than the soluble enzyme at elevated temperatures and to concentrated urea, and denaturation by urea at pH 8 is fully reversible since the loss of molecules by autolysis is eliminated.     

Catalytic and ligand binding properties of bovine trypsinogen and its complex with the effector dipeptide Ile-Val

Steady-state and pre-steady-state kinetic data for the trypsinogen catalyzed hydrolysis of a series of synthetic substrates (i.e. p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids) have been obtained as a function of pH (3.4-8). Moreover, the effect of ethylamine on the hydrolysis of a neutral substrate and benzamidine binding have been extensively studied. In order to obtain direct information on the transition of trypsinogen to a beta-trypsin-like structure, the role of the effector dipeptide Ile-Val on the catalytic and ligand binding properties of the zymogen has been investigated. Kinetic and thermodynamic data for beta-trypsin and alpha-chymotrypsin are also reported for the purpose of an homogeneous comparison of the various (pro)enzymes. Under all the experimental conditions, kinetic data for (pro)enzyme catalysis are consistent with the minimum three-step mechanism: (formula; see text) involving the acyl intermediate E X P. In the presence of Ile-Val dipeptide, trypsinogen assumes catalytic and ligand binding properties that are reminiscent of activated beta-trypsin. This is at variance with free trypsinogen, which shows a alpha-chymotrypsin-like behavior. The large differences in the results of kinetic and thermodynamic measurements for free trypsinogen, as compared to its binary adduct with Ile-Val, can be ascribed to the substantial differences in the two molecular species, which include the spatial orientation of Asp189.     

FORMATION OF TRYPSIN FROM CRYSTALLINE TRYPSINOGEN BY MEANS OF ENTEROKINASE

Crystalline trypsinogen is most readily and completely transformed into trypsin by means of enterokinase in the range of pH 5.2–6.0 at 5°C. and at a concentration of trypsinogen of not more than 0.1 mg. per ml. The action of enterokinase under these conditions is that of a typical enzyme. The process follows closely the course of a catalytic unimolecular reaction, the rate of formation of trypsin being proportional to the concentration of enterokinase added and the ultimate amount of trypsin formed being independent of the concentration of enterokinase. The catalytic action of enterokinase on crystalline trypsinogen in dilute solution at pH more alkaline than 6.0 and in concentrated solution at pH even slightly below 6.0 is complicated by the partial transformation of the trypsinogen into inert protein which can no longer be changed into trypsin even by a large excess of enterokinase. This secondary reaction is catalyzed by the trypsin formed and the rate of the reaction is proportional to the concentration of trypsin as well as to the concentration of trypsinogen in solution. Hence under these conditions only a small part of the trypsinogen is changed by enterokinase into trypsin while a considerable part of the trypsinogen is transformed into inert protein, the more so the lower the concentration of enterokinase used. The kinetics of the formation of trypsin by means of enterokinase when accompanied by the formation of inert protein can be explained quantitatively on the theoretical assumption that both reactions are of the simple catalytic unimolecular type, the catalyst being enterokinase in the first reaction and trypsin in the second reaction.     

Chymotrypsin C (caldecrin) stimulates autoactivation of human cationic trypsinogen

Trypsin-mediated trypsinogen activation (autoactivation) facilitates digestive zymogen activation in the duodenum but may precipitate pancreatitis if it occurs prematurely in the pancreas. Autoactivation of human cationic trypsinogen is inhibited by a repulsive electrostatic interaction between the unique Asp218 on the surface of cationic trypsin and the conserved tetra-aspartate (Asp19-22) motif in the trypsinogen activation peptide (Nemoda, Z., and Sahin-Tóth, M. (2005) J. Biol. Chem. 280, 29645-29652). Here we describe that this interaction is regulated by chymotrypsin C (caldecrin), which can specifically cleave the Phe18-Asp19 peptide bond in the trypsinogen activation peptide and remove the N-terminal tripeptide. In contrast, chymotrypsin B, elastase 2A, or elastase 3A (proteinase E) are ineffective. Autoactivation of N-terminally truncated cationic trypsinogen is stimulated approximately 3-fold, and this effect is dependent on the presence of Asp218. Because chymotrypsinogen C is activated by trypsin, and chymotrypsin C stimulates trypsinogen activation, these reactions establish a positive feedback mechanism in the digestive enzyme cascade of humans. Furthermore, inappropriate activation of chymotrypsinogen C in the pancreas may contribute to the development of pancreatitis. Consistent with this notion, the pancreatitis-associated mutation A16V in cationic trypsinogen increases the rate of chymotrypsin C-mediated processing of the activation peptide 4-fold and causes accelerated trypsinogen activation in vitro.     

Cathepsin B inhibition prevents trypsinogen activation and reduces pancreatitis severity

Intrapancreatic activation of trypsinogen is believed to play a critical role in the initiation of acute pancreatitis, but mechanisms responsible for intrapancreatic trypsinogen activation during pancreatitis have not been clearly defined. In previous in vitro studies, we have shown that intra-acinar cell activation of trypsinogen and acinar cell injury in response to supramaximal secretagogue stimulation could be prevented by the cell permeant cathepsin B inhibitor E64d (Saluja A, Donovan EA, Yamanaka K, Yamaguchi Y, Hofbauer B, and Steer ML. Gastroenterology 113: 304-310, 1997). The present studies evaluated the role of intrapancreatic trypsinogen activation, this time under in vivo conditions, in two models of pancreatitis by using another highly soluble cell permeant cathepsin B inhibitor, L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl-L-isoleucyl-L-proline methyl ester (CA-074me). Intravenous administration of CA-074me (10 mg/kg) before induction of either secretagogue-elicited pancreatitis in mice or duct infusion-elicited pancreatitis in rats markedly reduced the extent of intrapancreatic trypsinogen activation and substantially reduced the severity of both pancreatitis models. These observations support the hypothesis that, during the early stages of pancreatitis, trypsinogen activation in the pancreas is mediated by the lysosomal enzyme cathepsin B. Our findings also suggest that pharmacological interventions that inhibit cathepsin B may prove useful in preventing acute pancreatitis or reducing its severity.     

Crystal structure of enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide.

Enteropeptidase is a membrane-bound serine protease that initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. The enzyme is remarkably specific and cleaves after lysine residues of peptidyl substrates that resemble trypsinogen activation peptides such as Val-(Asp)4-Lys. To characterize the determinants of substrate specificity, we solved the crystal structure of the bovine enteropeptidase catalytic domain to 2.3 A resolution in complex with the inhibitor Val-(Asp)4-Lys-chloromethane. The catalytic mechanism and contacts with lysine at substrate position P1 are conserved with other trypsin-like serine proteases. However, the aspartyl residues at positions P2-P4 of the inhibitor interact with the enzyme surface mainly through salt bridges with the Nzeta atom of Lys99. Mutation of Lys99 to Ala, or acetylation with acetic anhydride, specifically prevented the cleavage of trypsinogen or Gly-(Asp)4-Lys-beta-naphthylamide and reduced the rate of inhibition by Val-(Asp)4-Lys-chloromethane 22 to 90-fold. For these reactions, Lys99 was calculated to account for 1.8 to 2.5 kcal mol(-1) of the free energy of transition state binding. Thus, a unique basic exosite on the enteropeptidase surface has evolved to facilitate the cleavage of its physiological substrate, trypsinogen.     

THE EFFECT OF CALCIUM AND OTHER IONS ON THE AUTOCATALYTIC FORMATION OF TRYPSIN FROM TRYPSINOGEN

Crystalline trypsinogen is completely transformed into trypsin by means of trypsin in the presence of calcium salts. The process follows the course of a pure autocatalytic unimolecular reaction. In the absence of calcium salts, the autocatalytic formation of trypsin from trypsinogen is complicated by the transformation of part of the trypsinogen into an inert protein which cannot be changed into trypsin by any known means. Salts increase or decrease the rate of both reactions so that the ultimate amount of trypsin formed varies with the nature and concentration of the salt used. With equivalent concentrations of salt the percentage of trypsinogen changed into trypsin is greatest in the presence of calcium ion followed in order by strontium; magnesium and sodium; rubidium, ammonium, lithium, and potassium; caesium and barium. With the anions the largest percentage of trypsinogen transformed into trypsin was found with the acetate, sulfate, oxalate, citrate, tartrate, fluoride, and chloride ions followed in order by bromide, nitrate, and iodide. The formation of inert protein is completely suppressed by concentrations of calcium ion greater than 0.02 M.     

In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules

The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.     

The energetic cost of induced fit catalysis: Crystal structures of trypsinogen mutants with enhanced activity and inhibitor affinity

The contribution of induced fit to enzyme specificity has been much debated, although with little experimental data. Here we probe the effect of induced fit on enzyme specificity using the trypsin(ogen) system. BPTI is known to induce trypsinogen to assume a trypsinlike conformation. Correlations are observed between BPTI affinity and the values of k(cat)/K(m) for the hydrolysis of two substrates by eight trypsin(ogen) variants. The slope of both correlations is -1.8. The crystal structures of the BPTI complexes of four variant trypsinogens were also solved. Three of these enzymes, K15A, DeltaI16V17/D194N, and DeltaI16V17/Q156K trypsinogen, are 10- to 100-fold more active than trypsinogen. The fourth variant, DeltaI16V17 trypsinogen, is the lone outlier in the correlations; its activity is lower than expected based on its affinity for BPTI. The S1 site and oxyanion hole, formed by segments 184A-194 and 216-223, are trypsinlike in all of the enzymes. These structural and kinetic data confirm that BPTI induces an active conformation in the trypsin(ogen) variants. Thus, changes in BPTI affinity monitor changes in the energetic cost of inducing a trypsinlike conformation. Although the S1 site and oxyanion hole are similar in all four variants, the N-terminal and autolysis loop (residues 142-152) segments have different interactions for each variant. These results indicate that zymogen activity is controlled by a simple conformational equilibrium between active and inactive conformations, and that the autolysis loop and N-terminal segments control this equilibrium. Together, these data illustrate that induced fit does not generally contribute to enzyme specificity.     

A common African polymorphism abolishes tyrosine sulfation of human anionic trypsinogen (PRSS2)

Human pancreatic trypsinogens undergo post-translational sulfation on Tyr(154), catalysed by the Golgi-resident enzyme tyrosylprotein sulfotransferase 2. Sequence alignments suggest that the sulfation of Tyr(154) is facilitated by a unique sequence context which is characteristically found in primate trypsinogens. In the search for genetic variants that might alter this sulfation motif, we identified a single nucleotide polymorphism (c.457G>C) in the PRSS2 (serine protease 2, human anionic trypsinogen) gene, which changed Asp(153) to a histidine residue (p.D153H). The p.D153H variant is common in subjects of African origin, with a minor allele frequency of 9.2%, whereas it is absent in subjects of European descent. We demonstrate that Asp(153) is the main determinant of tyrosine sulfation in anionic trypsinogen, as both the natural p.D153H variation and the p.D153N mutation result in a complete loss of trypsinogen sulfation. In contrast, mutation of Asp(156) and Glu(157) only slightly decrease tyrosine sulfation, whereas mutation of Gly(151) and Pro(155) has no effect. With respect to the biological relevance of the p.D153H variant, we found that tyrosine sulfation had no significant effect on the activation of anionic trypsinogen or the catalytic activity and inhibitor sensitivity of anionic trypsin. Taken together with previous studies, the observations of the present study suggest that the primary role of trypsinogen sulfation in humans is to stimulate autoactivation of PRSS1 (serine protease 1, human cationic trypsinogen), whereas the sulfation of anionic trypsinogen is unimportant for normal digestive physiology. As a result, the p.D153H polymorphism which eliminates this modification could become widespread in a healthy population.     

A simple method for isolating trypsin from trichloroacetic acid extracts of bovine pancreas

Trichloroacetic acid was used to isolate trypsin and trypsinogen from bovine pancreas. Trypsinogen, which is at first soluble in trichloroacetic acid, slowly forms a sediment. In alkaline medium and in the presence of calcium chloride, proenzyme is activated to enzyme which has high proteolytic, esterase, and amidase activity and is homogenous in polyacrylamide gel electrophoresis. It is suggested that the gradual reduction of trypsinogen solubility in trichloroacetic acid is associated with the presence of basic pancreatic trypsin inhibitor in trichloroacetic acid extracts from a bovine pancreas.     

Overexpression in Escherichia coli and functional reconstitution of anchovy trypsinogen from the bacterial inclusion body

We have synthesized and optimized a high-yielding Escherichia coli expression system to produce trypsinogen from anchovy Engraulis japonicus and have developed conditions for its successful refolding. Recombinant anchovy trypsinogen precipitated in E. coli Rosetta (DE3) placI strain as inclusion bodies was denatured by 6 M guanidine-HCl followed by refolding with drop wise addition to a large excess of a folding buffer containing 0.5 M non-detergent sulfobetaine (NDSB-251) and a redox potential of oxidized and reduced glutathiones. The folded trypsinogen was autocatalytically activated to its mature form, trypsin, and purified with a MonoQ ion-exchange column. NH₂-terminal amino acid sequencings revealed that E. coli efficiently processed NH₂-terminal methionine residue from the expressed trypsinogen and that trypsinogen was activated at the correct site to generate active trypsin. The recombinant enzyme showed kinetic properties comparable to those of the native enzyme and demonstrated a typical cleavage preference for arginine over lysine residue against a protein substrate. The optimized expression and folding procedures yielded 12 mg of purified, active trypsin from 1 L of bacterial culture or 45 g wet weight cells, which is quite enough for various analytical and semipreparative purposes.     

金鱼胰蛋白酶原基因的克隆及镉和过氧化氢暴露对其基因表达的影响

为深入研究胰蛋白酶在鱼类中的蛋白结构和生理功能, 利用RT-PCR和RACE方法, 从金鱼肝胰脏中成功克隆获得了一种全长864 bp的胰蛋白酶原cDNA序列(gfTryp)。gfTrypc DNA包含21 bp的5′-非翻译区、114 bp的3′-非翻译区和729 bp的开放读码框, 编码242个氨基酸组成的胰蛋白酶原(gfTryp)。gfTryp含有15个氨基酸的信号肽和5个氨基酸(LDDDK)的激活肽。氨基酸序列分析表明, gfTryp具备胰蛋白酶原的保守结构特征, 如含有催化三联体氨基酸(His-57、Asp-102和Ser-195), 12个半胱氨酸, 位于底物结合口袋底部Asp-189和口袋开口处的Gly-216、Gly-226等, 提示其可能具有保守的蛋白消化功能。RT-PCR结果显示, gfTryp mRNA在所检测的各个组织中均有表达, 其中在肝胰脏、肠和脂肪中表达量为最高。进一步研究发现, 相较于摄食前, 肝胰脏gfTryp mRNA在金鱼摄食后显著升高。在0.5和5 μg/mL镉暴露处理后, 肝胰脏gfTryp mRNA显著升高(与未处理组相比, 分别约为3.2 和 4.7倍)。随着镉浓度增加到10 μg/mL后, gfTryp mRNA表达量下降。经100 μmol/L过氧化氢处理3h、6h、12h和24h后, 金鱼肝胰脏gfTryp mRNA的表达水平均显著下降, 在6h达到最大效应(约为对照组的0.21倍)。研究结果证实了重金属镉和过氧化氢处理能调控胰蛋白酶原基因表达, 为进一步探讨鱼类消化生理提供了新的视角。     

Autoactivation of Mouse Trypsinogens Is Regulated by Chymotrypsin C via Cleavage of the Autolysis Loop

Chymotrypsin C (CTRC) is a proteolytic regulator of trypsinogen autoactivation in humans. CTRC cleavage of the trypsinogen activation peptide stimulates autoactivation, whereas cleavage of the calcium binding loop promotes trypsinogen degradation. Trypsinogen mutations that alter these regulatory cleavages lead to increased intrapancreatic trypsinogen activation and cause hereditary pancreatitis. The aim of this study was to characterize the regulation of autoactivation of mouse trypsinogens by mouse Ctrc. We found that the mouse pancreas expresses four trypsinogen isoforms to high levels, T7, T8, T9, and T20. Only the T7 activation peptide was cleaved by mouse Ctrc, causing negligible stimulation of autoactivation. Surprisingly, mouse Ctrc poorly cleaved the calcium binding loop in all mouse trypsinogens. In contrast, mouse Ctrc readily cleaved the Phe-150–Gly-151 peptide bond in the autolysis loop of T8 and T9 and inhibited autoactivation. Mouse chymotrypsin B also cleaved the same peptide bond but was 7-fold slower. T7 was less sensitive to chymotryptic regulation, which involved slow cleavage of the Leu-149–Ser-150 peptide bond in the autolysis loop. Modeling indicated steric proximity of the autolysis loop and the activation peptide in trypsinogen, suggesting the cleaved autolysis loop may directly interfere with activation. We conclude that autoactivation of mouse trypsinogens is under the control of mouse Ctrc with some notable differences from the human situation. Thus, cleavage of the trypsinogen activation peptide or the calcium binding loop by Ctrc is unimportant. Instead, inhibition of autoactivation via cleavage of the autolysis loop is the dominant mechanism that can mitigate intrapancreatic trypsinogen activation.     

Comparative studies on the mechanism of activation of the two human trypsinogens

The activation of human trypsinogens 1 and 2 by porcine enterokinase at pH 5.6 shows that the two human zymogens are equivalent substrates for this enzyme and that both proteins are activated faster than the cationic bovine trypsinogen. At pH 8.0 and in the presence of 20 mM calcium the two human trypsinogens are activated by either human trypsin at the same rate but the affinity of both trypsins is higher for trypsinogen 1 than for trypsinogen 2. Two Ca2+ binding sites are identified in the two human zymogens and their pK(Ca2+) values determined. For trypsinogen 1 the values are respectively of 2.8 and 3.3 for the primary and secondary Ca2+ binding sites, and for trypsinogen 2 of 3.4 and 2.7. These values are markedly different from those obtained for bovine cationic trypsinogen, especially in the case of trypsinogen 1. These results point out a different degree of saturation of the calcium binding sites of the 2 human zymogens that must exist in physiological conditions, suggesting different biological activities of the two trypsinogens.     

Unusual kinetic behavior of Endothia parasitica protease in hydrolysis of small peptides

Endothia parasitica protease hydrolyzes l-leucyl-l-leucine amide and l-leucyl-l-phenylalanine amide at the peptide bond. l-Phenylalanyl-l-leticine amide, N-carbobenzoxy-l-leucyl-l-phenylalanine amide, N-carbobenzoxy-l-leucyl-l-pheml-alanine, N-carbobenzoxy-l-phenylalanyl-l-valine amide, and l-leucyl-β-naphthyl-amide are not hydrolyzed. In contrast to the kinetics of hydrolysis of casein and oxidized B-chain of insulin and activation of trypsinogen by Endothia parasitica protease which are normal, reaction progress curves for hydrolysis of l-leucyl-l-leucine amide and l-leucyl-l-phenylalanine amide are sigrnoidal. Initially, the reaction rates were of the order of 0.5–2.5% of the maximum rates eventually attained. With increasing time of incubation the reaction rates became faster and faster until maximum rates were achieved. This abnormal behavior was not eliminated by recrystallization of substrate or by incubation of enzyme alone or with products of the reaction prior to addition of substrate. Addition of a new aliquot of substrate, vizl-leucyl-l-leucine amide, to the reaction prior to complete hydrolysis of all of a previous aliquot of the same substrate, or reactions containing a mixture of oxidized B chain of insulin and l-leucyl-l-leucine amide, gave normal reaction progress curves. The duration of abnormal behavior before a maximum rate was attained was a function of enzyme concentration and temperature but not of substrate concentration even though substrate was in less than saturating amounts. The reaction data follow second-order autocatalytic kinetics with respect to enzyme concentration. It is proposed that most of the enzyme is in an inactive form in absence of substrate but is rapidly converted to the active form on combination with a good substrate such as trypsinogen, casein, or oxidized B chain of insulin. However, with a poor substrate such as l-leucyl-l-leucine amide, conversion to active enzyme is mediated through formation of an active enzyme-inactive enzyme complex followed by combination with substrate and hydrolysis.     

Biochemical changes in the exocrine pancreas of rats fed caffeine

Coffee consumption has been associated with pancreatic disorders, but the mechanisms involved remain to be elucidated. This investigation examines the effects of caffeine consumption on the structure and function of the exocrine pancreas. Groups of rats, fed ad libitum commercial laboratory diet, were given drinking water which contained either caffeine (0.09 mg/ml) or nothing at all. The rats were allowed drink ad libitum and were killed 6 weeks later. Final body and pancreatic weights were not significantly different between the groups at the end of the experimental period. Although no ultrastructural effects of caffeine on the pancreas were observed, amylase and trypsinogen activity was 35% higher in pancreatic homogenates from caffeine-fed rats compared with controls. In addition, levels of immunoreactive cationic trypsin(ogen) were 41% higher than control levels in pancreases from the caffeine-fed rats. Also, the circulating levels of amylase and immunoreactive cationic trypsin(ogen) in serum were lower in the caffeine group compared with controls. When dispersed pancreatic acini isolated from the caffeine-fed rats were incubated in vitro with increasing concentrations of CCK-8 or nicotine, the rate of release of amylase, trypsinogen, and chymotrypsinogen was lower than in the control rats. This effect did not appear to be due to inhibition of protein synthesis, as determined by [3H]leucine incorporation into acinar protein. These data suggest that prolonged intake of caffeine at common dietary levels inhibits pancreatic enzyme secretion.     

Differential increases in syntheses of newly identified trypsinogen 2 isoforms by dietary protein in rat pancreas

We have found that dietary protein markedly induced pancreatic serine protease activity via a mechanism independent of luminal trypsin activity in pancreaticobiliary-diverted (PBD) rats. The aim of this study was to examine the effects of dietary protein on the synthesis of trypsinogen isoforms by comparing in vivo incorporation of [35S] L-methionine into isoform proteins in PBD and sham-operated rats. A small duodenal segment including the ampulla of Vater was sectioned and transposed to the upper ileum with end-to-side anastomosis (PBD) or duodenal transection was followed by reanastomosis (sham) in male Sprague-Dawley rats. After recovery, PBD and sham rats were fed a 25% or 60% casein-sucrose-based diet (NC or HC) for 14 days. Rats were then intravenously injected with [35S] L-methionine (15 MBq/kg body weight) 30 mins before being sacrificed for analysis of pancreatic enzymes by two-dimensional SDS-polyacrylamide gel electrophoresis. By using electrophoresis with narrow range of isoelectric focusing (pI 4.5-5.5), five trypsinogen 2 (2-x) isoform spots were identified using both [35S] incorporation and Coomassie brilliant blue (CBB) staining in PBD rats, but not in sham rats. N-terminal sequences of these trypsinogen 2-x spots were identical to known rat trypsinogen 2 with the exception that the third valine was changed to isoleucine in one isoform. In PBD rats, feeding of HC specifically increased the [35S] and CBB intensities of these trypsinogen 2-x isoforms and trypsinogen 3. The degree of induction of the five trypsinogen 2-x molecules by HC varied greatly. Trypsinogen 1 and 4, which are the major trypsinogens in normal rats, showed no changes. We conclude that increases in synthesis of a few newly identified trypsinogen 2-x isoforms mainly contribute to the induction of trypsin activity in the pancreas by HC in PBD rats.     

Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin

To test the role of Asp-189 which is located at the base of the substrate binding pocket in determining the specificity of trypsin toward basic substrates, this residue was replaced with a lysine residue by site-directed mutagenesis. Both rat trypsinogen and Lys-189 trypsinogen were expressed and secreted into the periplasmic space of Escherichia coli. The proteins were purified to homogeneity and activated by porcine enterokinase, and their catalytic activities were determined on natural and synthetic substrates. Lys-189 trypsin displayed no catalytic activity toward arginyl and lysyl substrates. Further, there was no compensatory change in specificity toward acidic substrates; no cleavage of aspartyl or glutamyl bonds was detected. Additional studies of substrate specificity involving gas-phase sequence analyses of digested natural substrates revealed an inherent but low chymotrypsin-like activity of trypsin. This activity was retained but modified by the Asp to Lys change at position 189. In addition to hydrolyzing phenylalanyl and tyrosyl peptide bonds, the mutant enzyme has the unique property of cleaving leucyl bonds. On the basis of computer graphic modeling studies of the Lys-189 side chain, it appears that the positively charged NH2 group is directed outside the substrate binding pocket. The resulting hydrophobic cavity may explain the altered substrate specificity of the mutant enzyme. The relatively low chymotrypsin-like activity of both recombinant enzymes may be due to distorted positioning of the scissile bond with respect to the catalytic triad rather than to the lack of sufficient interaction between the hydrophobic side chains and the substrate binding pocket of the enzyme.