Rat Serum Factors Inhibiting the G1-S Transition In Hepatocytes

An attempt was made to detect the serum factors inhibiting the G₁-S transition in synchronized, baby rat hepatocytes. In untreated adult rat serum, this inhibitory activity was always linked to high molecular weight (HMW) compounds. Incubation of serum with trypsin or chymotrypsin resulted in the formation of a low molecular weight (LMW) G₁-S inhibitory factor. the same result was obtained with fractions from adult rat liver but not with kidney or spleen fractions. Separation of the LMW factor by ultrafiltration increased its specific activity by about 10³. the active period in the cell cycle of both the LMW and HMW factors was the same: the late G₁ phase. However, the activity of the LMW factor was not blocked by the Kunitz factor. an enzymatic transformation of the HMW factor might be induced by liver cell membrane-bound proteases and constitute a mechanism regulating hepatocyte proliferation.     

Isolation of tissue kallikrein in rat spleen by monoclonal antibody-affinity chromatography

Rat spleen kallikrein was identified and purified by DEAE-cellulose and monoclonal antibody-affinity chromatography. The purified enzyme has Tos-Arg-OMe esterase activity and kinin-releasing activity from a purified low-molecular-weight kininogen substrate. In the direct radioimmunoassay for tissue kallikrein, the splenic enzyme displays parallelism with standard curves of rat urinary kallikrein. The pH profiles of the Tos-Arg-OMe esterase activities of spleen and urinary kallikrein were identical with optima at 9.0 Rat spleen kallikrein was inhibited strongly by aprotinin and affinity-purified kallikrein antibody and weakly by soybean trypsin inhibitor. The IC₅₀ values were similar to those observed against rat urinary kallikrein. Neither the urinary nor the splenic enzyme was inhibited by lima bean trypsin inhibitor or preimmune serum immunoglobulins. Spleen kallikrein was labeled with [¹⁴]diisopropylphosphorofluoridate and visualized by fluorography on a sodium dodecyl sulfate-polyacrylamide gel. The electrophoretic mobility of the splenic enzyme was indistinguishable from that of urinary kallikrein A with an estimated Mr of approx. 38 000. With Western blot analyses using a rabbit anti-kallikrein antibody followed by ¹²⁵I-labeled protein A binding, the spleen and urinary kallikreins were again visualized at identical positions by autoradiography. The data show that there is a rat splenic tissue kallikrein which is indistinguishable from a renal kallikrein with respect to physicochemical properties, immunological character and susceptibility to inhibitors.     

Activation of delta5-3-ketosteroid isomerase of bovine adrenal microsomes by serum albumins.

The Δ⁵-3-ketosteroid isomerase (EC 5.3.3.1) of bovine adrenal microsomes is activated as much as 10- to 20-fold by micromolar concentrations of bovine serum albumin. Comparable activations are observed with the serum albumins of 10 other mammalian species, but are not seen with ovalbumin or conalbumin. Evidence that the activation is attributable to the serum albumins, rather than to a small, firmly-bound ligand, is based on: (1) Failure to remove the stimulatory activity from the albumin by chloroform extraction, dialysis, or gel filtration; (2) Destruction of the activity by heating or by trypsin digestion; (3) Precipitation of the stimulatory activity of bovine serum albumin by specific antibody. Bovine serum albumin induces small decreases in the Michaelis constant for Δ⁵-androstene-3,17-dione, but most of the activational effect reflects an increase in the maximum velocity. Low concentrations of Triton X-100, which are without effect on the isomerase activity, prevent the activation by bovine serum albumin.     

Preparation, properties, and uses of two fluorogenic substrates for the detection of 5'-(venom) and 3'-(spleen) nucleotide phosphodiesterases

A new method for ³H-labeling of native collagen and a specific microassay for collagenase activity are presented. Acid-soluble type I collagen derived from rat tail tendons was reacted with pyridoxal phosphate and then reduced with NaB³H₄ to yield [³H]collagen with a specific activity of more than 10 μCi/mg. With respect to rate of hydrolysis, trypsin susceptibility, and gelling properties this collagen compares favorably with biosynthetically labeled preparations. It was shown that chemical labeling procedures such as this, or N-acetylation with acetic anhydride, do not adversely affect properties of collagen which are important for its use as substrate in specific assays. The microassay employs 50-μl [³H]collagen gels (1 mg/ml) dispensed in microtest plates. At 36°C this assay combines rapid rate of hydrolysis with low trypsin susceptibility. As little as 1 ng of clostridial collagenase activity can be measured reproducibly. The high specific activity of the [³H]collagen allowed us to explore microassay conditions employing minute quantities of substrate in solution. These studies indicated that native type I collagen whether labeled or not, is cleaved in the helical region by trypsin at subdenaturation temperatures. It was concluded that, in order to remain specific, collagenase assays with collagen in solution as with collagen in fibrils must be performed at 10–12°C below the denaturation temperature, i.e., at 35–37°C with collagen gels and 27–29°C with collagen in solution.     

Proteases as mitogens : The effect of trypsin and pronase on mouse and human lymphocytes

Treatment of mouse spleen lymphocytes with trypsin (from 0.1 to 1.0 μg/ml) was found to cause a significant stimulation of the incorporation of ³H-thymidine. When spleen cells from nude (congenitally athymic) mice were incubated with trypsin in the absence of serum for 3 days, very high levels of incorporation were noted (stimulation index of 10 to 20). Trypsin was without effect on thymic lymphocytes of the mouse but caused significant activation of human peripheral blood lymphocytes. The stimulatory effect of trypsin was a consequence of its enzymatic activity. Prolonged treatment with pronase also caused small but significant increases in the incorporation of labelled thymidine (stimulation index of 2 to 4) into the thymic and splenic lymphocytes of the mouse and into human lymphocytes. The evidence suggests that trypsin stimulates the B-derived lymphocytes.     

Effect of protease inhibitors on mitogen stimulation of hamster lymphoid cells

Hamster lymph node and spleen cells can be stimulated to incorporate tritiated thymidine ([³H]TdR) in vitro under serum-free conditions by the proteases trypsin and chymotrypsin. Under similar conditions, thymocytes could be stimulated by concanavalin A (ConA) but not lipopolysaccharide (LPS) or the proteases. The subpopulation of cells responding to the proteases correlated with the cells responding to LPS on fractionation of spleen and lymph node cells on discontinuous bovine serum albumin (BSA) gradients or on nylon-wool columns. The stimulation induced by trypsin was completely blocked by soybean trypsin inhibitor (SBTI) while that induced by chymotrypsin was only partially blocked. The inhibition by SBTI of protease activation was not effective when added 24 h after initiation of stimulation. On the other hand, addition of clarified isologous serum to protease activated cultures after 24 h still lead to greater than 50% inhibition of [³H]TdR incorporation.     

THE ANTIPROTEOLYTIC ACTIVITY OF SERUM : I. THE NATURE AND EXPERIMENTAL VARIATION OF THE ANTIPROTEOLYTIC ACTIVITY OF SERUM

1. An equation is derived for the calculation of a constant which, experimental results indicate, may be a more reliable index of the antiproteolytic activity of serum than those equations hitherto used. 2. (a) Intramuscular administration of trypsin resulted in a slow rise in the antiproteolytic activity of the serum, followed by a lesser decline. (b) Intravenous administration resulted in no appreciable variation. (c) Oral administration resulted in a rapid rise, which was sustained during the period of administration. (d) Intramuscular, intravenous, or oral administration of denatured trypsin resulted in no appreciable variation. (e) The extent of the local necrosis following subcutaneous injection of trypsin varied inversely with the antiproteolytic activity of the serum. 3. The experimental results indicate that the products of protein hydrolysis in the intestine and parenterally are an important factor in the antiproteolytic activity of the serum. They also indicate that antibodies to trypsin are not an important factor in the antiproteolytic activity of the serum.     

Purification and Characterization of Albumin from Frog Skin of <Emphasis Type="Italic">Duttaphrynus melanostictus</Emphasis>

Following determination of trypsin inhibitory activity, a serine protease inhibitor was purified and characterized from frog Duttaphrynus melanostictus serum. It was identified as serum albumin, with molecular weight of 67 kDa (DmA-serum). Different from bovine serum albumin, DmA-serum potently inhibited trypsin with similar K _i values around 1.6 × 10⁻⁷ M. No inhibitory effect on thrombin, chymotrypsin, elastase and subtilisin was observed under the assay conditions. The N-terminal amino acid is EAEPHSRI. Subsequently, a protein with same N-terminal amino acid was purified from skin, termed as DmA-skin. However, DmA-skin is distinct from DmA-serum by binding of a haem b (0.5 mol/mol protein), and with low trypsin inhibitory activity. Frog albumin is distributed in frog skin and exhibited trypsin inhibitory activity, suggesting that it plays important roles in skin physiological functions, like water economy, metabolite exchange and osmoregulation, etc.     

Relative stability of α-melanotropin and related analogues to rat brain homogenates

α-Melanotropin (α-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle⁴, D-Phe⁷]-α-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle⁴, D-Phe⁷]-α-MSH_4–10-NH₂, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle⁴, D-Phe⁷]-α-MSH_4–11-NH₂ is totally resistant to inactivation by rat brain homogenate. Both [Nle⁴, D-Phe⁷]-fragments are resistant to degradation by rat serum, but [Nle⁴]-α-MSH, Ac-[Nle⁴]-α-MSH_4–10-NH₂ and Ac-[Nle⁴]-α-MSH_4–11-NH₂ are rapidly inactivated under both conditions. The cyclic melanotropin, [ ]-α-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[ ]-α-MSH_4–10-NH₂ analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[ ]-α-MSH_4–10-NH₂ is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.     

Gastrointestinal Transit of Soybean Trypsin Inhibitor and Fluctuations in Intraluminal Trypsin

Soybean trypsin inhibitor of Kunitz type (STI) was modified by reduced alkylation with NaBH₄ and HCHO, and examined for resistance to in vitro digestion from the viewpoint of inhibitory activity and immunoreactivity. Methylated STI was exactly alike in that respect. Hence, STI was in part replaced by its [¹⁴C]methyl-labeled specimen and their (17: 3) mixture was used as a proteinous and unabsorbable marker for digestibility experiments. When STI was immunochemically measured in intraluminal leavings in segments of the rat digestive tract, its recovery decreased progressively with the elapse of time. As a result of radioactivity measurement, however, the recovery of [¹⁴C]labeled STI proved almost quantitative all together over a period of 7 h postprandial. Concurrently, gastric emptying and intestinal transit of digesta were assessed in terms of intraluminal STI movement. After moving out from the stomach, STI rapidly passed through the upper small intestine with depression of the trypsin activity, and stayed in the lower small intestine for a few or several hours (all that while, the trypsin activity was not depressed so much). A similar pattern was observed for the intraluminal movement of a food additive ‘indigo carmine’ in another experiment. It was assumed from these observations that digesta would also have gone past the upper small bowel irrespective of ingesting either a powdered 20% casein diet or a dumpling-kneaded artificial bait.     

Stimulation of yeast mitochondrial protein synthesis by postpolysomal supernatants from yeast,rat liver,and Escherichia coli

Isolated yeast mitochondria incubated with a protein-synthesizing mixture containing excess oxidizable substrate, amino acids, MgCl₂, an ATP-regenerating system, and optimal levels of [³H]leucine cease protein synthesis after 30 min. Postpolysomal supernatants from either yeast, rat liver, or Escherichia coli can restore protein synthetic activity to depleted yeast mitochondria; however the addition of bovine serum albumin to the incubation mixture did not restore activity. The restored incorporation activity was sensitive to chloramphenicol, insensitive to cycloheximide, and proportional to the protein concentration of the supernatants. Furthermore, addition of all three high-speed supernatants to isolated mitochondria at time zero stimulated the rate of protein synthesis to a greater extent than when these fractions were added to depleted mitochondria. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the translation products obtained from mitochondria labeled in vitro in the presence of supernatant fractions were identical to the proteins labeled by mitochondria in vivo; however, the synthesis of the bands corresponding to subunit III of cytochrome oxidase, cytochrome b, and VAR-3 was stimulated to the greatest extent. The stimulatory activity in the supernatants was non-dialyzable, insensitive to treatment with ribonuclease A, but completely abolished by pretreatment with trypsin suggesting that the stimulatory factor(s) is of a protein nature. The postpolysomal supernatants did not incorporate amino acids into protein when incubated without mitochondria. These results suggest that the protein synthetic capacity of mitochondria is apparently limited by extramitochondrial proteins which are present in either yeast, rat liver, or E. coli.     

Substrate activation of rat trypsins 1 and 2

Qualitative differences in the active center of rat trypsins 1 and 2 resulted in different ratios of K_cat for N¹-tosyl-l-arginine methyl ester vs K_cat for N¹-benzoyl-l-arginine ethyl ester. These ratios were 2.5 for trypsin 1 and 1.2 for trypsin 2.Substrate activation with N¹-tosyl-l-arginine methyl ester enhanced the catalytic rate constant of rat trypsin 1 2.5-fold and that of rat trypsin 2 only 1.5-fold. The increase in the catalytic rate constant found with N¹-benzoyl-l-arginine ethyl ester was the same (1.5-fold) for both trypsins. Consequently, at 20 mm substrate concentration, trypsin 1 catalyzed the esterolysis of N¹-tosyl-l-arginine methyl ester 4.5 times faster than that of N¹-benzoyl-l-arginine ethyl ester, while trypsin 2 was only 1.3 times more efficient with the first substrate.Furthermore, the activation of both rat enzymes by N-acetyl-l-tyrosine ethyl ester was even more effective than that obtained with the two cationic esters; the maximum rates of hydrolysis of this neutral substrate by trypsins 1 and 2 were enhanced 120- and 50-fold, respectively, by high concentrations of N-acetyl-l-tyrosine ethyl ester.     

Trypsin complexed with α1-proteinase inhibitor has an increased structural flexibility

Mutant rat trypsin Asp¹⁸⁹Ser was prepared and complexed with highly purified human α₁-proteinase inhibitor. The complex formed was purified to homogeneity and studied by N-terminal amino acid sequence analysis and limited proteolysis with bovine trypsin. As compared to uncomplexed mutant trypsin, the mutant enzyme complexed with α₁-proteinase inhibitor showed a highly increased susceptibility to enzymatic digestion. The peptide bond selectively attacked by bovine trypsin was identified as the Arg¹¹⁷-Val¹¹⁸ one of trypsin. The structural and mechanistic relevance of this observation to serine proteinase-substrate and serine proteinase-serpin reactions are discussed.     

On the presence of a nicotinamide nucleotide transhydrogenase in mitochondria from potato tuber

Mitochondria isolated from potato (Solanum tuberosum L.) tuber were investigated for the presence of a nicotinamide nucleotide transhydrogenase activity. Submitochondrial particles derived from these mitochondria by sonication catalyzed a reduction of NAD⁺ or 3-acetylpyridine-NAD⁺ by NADPH, which showed a maximum of about 50 to 150 nanomoles/minute·milligram protein at pH 5 to 6. The K_m values for 3-acetylpyridine-NAD⁺ and NADPH were about 24 and 55 micromolar, respectively. Intact mitochondria showed a negligible activity in the absence of detergents. However, in the presence of detergents the specific activity approached about 30% of that seen with submitochondrial particles. The potato mitochondria transhydrogenase activity was sensitive to trypsin and phenylarsine oxide, both agents that are known to inhibit the mammalian transhydrogenase. Antibodies raised against rat liver transhydrogenase crossreacted with two peptides in potato tuber mitochondrial membranes with a molecular mass of 100 to 115 kilodaltons. The observed transhydrogenase activities may be due to an unspecific activity of dehydrogenases and/or to a genuine transhydrogenase. The activity contributions by NADH dehydrogenases and transhydrogenase to the total transhydrogenase activity were investigated by determining their relative sensitivities to trypsin. It is concluded that, at high or neutral pH, the observed transhydrogenase activity in potato tuber submitochondrial particles is due to the presence of a genuine and specific high molecular weight transhydrogenase. At low pH an unspecific reaction of an NADH dehydrogenase with NADPH contributes to the total trans-hydrogenase activity.     

Oostatic hormone inhibits biosynthesis of midgut proteolytic enzymes and egg development in mosquitoes

Injection of partially purified oostatic hormone (0.7 μg) into female Aedes aegypti inhibited egg development, proteolytic enzyme activity, and blood digestion in the midgut, whereas control injections of saline or insulin chain A (0.7 μg) did not affect these processes. Oostatic hormone given by enema, on the other hand, did not inhibit proteolytic enzyme activity, indicating that the hormone acts outside the midgut. A single injection of oostatic hormone (0.7 μg) caused a 1.7–1.5-fold reduction in activity of trypsinlike enzymes during blood digestion, with a 10-h delay in peak activity. Using [1,3-³H]diisopropylfluorophosphate (DFP) in the presence of 8 mM tosylamide-2-phenylethyl chloromethyl ketone, the synthesis of trypsinlike derivatives was followed in the midgut of female A. aegypti. A 4-fold reduction in [1,3-³H]diisopropylphosphoryl-trypsinlike derivatives was noted after oostatic hormone treatment. Several isozymes that are normally synthesized were absent in the presence of DFP, as assessed by polyacrylamide gel electrophoresis. Injection of oostatic hormone into decapitated and ovariectomized females that did not synthesize ecdysteroids inhibited trypsinlike enzyme synthesis and blood digestion in the midgut, indicating that oostatic hormone inhibits the midgut cells and not the ovary or the brain's endocrine system. Comparison between oostatic hormone and soybean trypsin inhibitor indicated that the former inhibited trypsin synthesis whereas the latter inhibited trypsin activity. A. aegypti oostatic hormone is not species specific and injections of the hormone into Culex quinquefasciatus, Culex nigripalpus, and Anopheles albimanus caused inhibition of egg development, blood digestion, and synthesis of trypsinlike enzymes. A direct relation between oostatic hormone synthesis and the regulation of trypsinlike activity in the midgut is proposed.     

Mechanism and Nature of Inhibition of Trypsin by Ligupurpuroside A,a Ku-Ding Tea Extract,Studied by Spectroscopic and Docking Methods

Ligupurpuroside A is a glycoside extracted from Ku-Ding tea. As extracts from Ku-Ding tea exhibit anti-inflammatory property, we hypothesize that Ligupurpuroside A may be an active compound which inhibits trypsin activity during the anti-inflammatory process. The mechanism and nature of inhibition of trypsin by Ligupurpuroside A have been studied by multi-spectroscopic method, enzyme-activity assay and molecular docking. Enzyme activity assay reveals that Ligupurpuroside A significantly inhibits the activity of trypsin through a competitive manner with an IC₅₀ value of 3.08 × 10^?3 mol L^?1. Fluorescence titration together with thermodynamic analysis indicate that a Ligupurpuroside A-trypsin complex is formed, and that hydrophobic force and hydrogen bonding are the main forces stabilizing the complex. UV-vis absorption, synchronous fluorescence and circular dichroism spectra show that the interaction between Ligupurpuroside A and trypsin induces conformational changes of trypsin with a decrease in the contents of α-helix and β-sheet. Finally, molecular docking further suggests that Ligupurpuroside A molecule binds within the active pocket of trypsin via hydrophobic force and hydrogen bond. Results from this study of the interaction of trypsin with its natural inhibitor should be useful to minimize the antinutritional effects and make full use of tea extracts in the food industry, and be also helpful to the design of the drugs for the diseases related to overexpression of trypsin.     

Renal cortical basolateral Na+/HCO 3 − cotransporter III. Evidence for a regulatory protein in the inhibitory effect of protein kinase A

The activity of the Na-H antiporter is inhibited by cyclic AMP-dependent protein kinase A (cAMP.PKA). The inhibitory effect of PKA on the Na-H antiporter is mediated through a regulatory protein that can be dissociated from the antiporter by limited protein digestion. PKA also inhibits the activity of the Na⁺/ HCO ₃ ^? cotransporter. We investigated whether the activity of Na⁺/HCO ₃ ^? cotransporter and the effect of PKA on this transporter may also be regulated by limited protein digestion. In rabbit renal cortical basolateral membranes (BLM) and in solubilized BLM reconstituted in liposomes (proteoliposomes), trypsin (100 μg) increased ²²Na uptake in the presence of HCO₃ but not in the presence of gluconate, indicating that trypsin does not alter diffusive ²²Na uptake but directly stimulates the Na⁺/HCO ₃ ^? cotransporter activity. In proteoliposomes phosphorylated with ATP, the catalytic subunit (CSU) of cAMP-PKA decreased the activity of the Na⁺/HCO ₃ ^? cotransporter (expressed as nanomoles/mg protein/3s) from 23 ± 10 to 14 ± 6 (P < 0.01). In the presence of trypsin, the inhibitory effect of CSU of cAMP-PKA on the activity of Na⁺/HCO ₃ ^? cotransporter was blunted. To identify a fraction that was responsible for the inhibitory effect of the CSU on the Na⁺/HCO ₃ ^? cotransporter activity, solubilized proteins were separated by size exclusion chromatography. The effect of CSU of cAMP-PKA on the Na⁺/HCO ₃ ^? cotransporter activity was assayed in proteoliposomes digested with trypsin with the addition of a fraction containing the 42 kDa protein (fraction S+) or without the 42 kDa protein (fraction S?). With the addition of fraction S?, the CSU of cAMP-PKA failed to inhibit the Na⁺/HCO ₃ ^? cotransporter activity (control 27 ± 6, CSU 27 ± 3) while the addition of fraction S+ restored the inhibitory effect of CSU (27 ± 6 to 3 ± 0.3 P < 0.01). The CSU of cAMP-PKA phosphorylated several proteins in solubilized protein including a 42 kDa protein. Fluorescein isothiocyanate (FITC) labels components of the Na⁺/HCO ₃ ^? cotransporter including the 56 kDa and 42 kDa proteins. In trypsin-treated solubilized protein the 42 kDa protein was not identified with FITC labeling. The results demonstrate that the activity of the Na⁺/HCO ₃ ^? cotransporter is regulated by protein(s) which mediates the inhibitory effect of PKA. Limited protein digestion can dissociate this protein from the cotransporter.     

Age-Dependent Effects of UCP2 Deficiency on Experimental Acute Pancreatitis in Mice

Reactive oxygen species (ROS) have been implicated in the pathogenesis of acute pancreatitis (AP) for many years but experimental evidence is still limited. Uncoupling protein 2 (UCP2)-deficient mice are an accepted model of age-related oxidative stress. Here, we have analysed how UCP2 deficiency affects the severity of experimental AP in young and older mice (3 and 12 months old, respectively) triggered by up to 7 injections of the secretagogue cerulein (50 μg/kg body weight) at hourly intervals. Disease severity was assessed at time points from 3 hours to 7 days based on pancreatic histopathology, serum levels of alpha-amylase, intrapancreatic trypsin activation and levels of myeloperoxidase (MPO) in lung and pancreatic tissue. Furthermore, in vitro studies with pancreatic acini were performed. At an age of 3 months, UCP2^-/- mice and wild-type (WT) C57BL/6 mice were virtually indistinguishable with respect to disease severity. In contrast, 12 months old UCP2^-/- mice developed a more severe pancreatic damage than WT mice at late time points after the induction of AP (24 h and 7 days, respectively), suggesting retarded regeneration. Furthermore, a higher peak level of alpha-amylase activity and gradually increased MPO levels in pancreatic and lung tissue were observed in UCP2^-/- mice. Interestingly, intrapancreatic trypsin activities (in vivo studies) and intraacinar trypsin and elastase activation in response to cerulein treatment (in vitro studies) were not enhanced but even diminished in the knockout strain. Finally, UCP2^-/- mice displayed a diminished ratio of reduced and oxidized glutathione in serum but no increased ROS levels in pancreatic acini. Together, our data indicate an aggravating effect of UCP2 deficiency on the severity of experimental AP in older but not in young mice. We suggest that increased severity of AP in 12 months old UCP2^-/- is caused by an imbalanced inflammatory response but is unrelated to acinar cell functions.     

Functional expression of trypsin from Streptomyces griseus by Pichia pastoris

In the present study, the genes encoding trypsinogen and active trypsin from Streptomyces griseus were both cloned and expressed in the methylotrophic yeast Pichia pastoris with the α-factor secretion signal under the control of the alcohol oxidase promoter. The mature trypsin was successfully accumulated extracellularly in soluble form with a maximum amidase activity of 6.6?U?ml^?1 (batch cultivation with flask cultivation) or 14.4?U?ml^?1 (fed-batch cultivation with a 3-l fermentor). In contrast, the recombinant trypsinogen formed inclusion bodies and no activity was detected. Replacement of the trypsin propeptide Ala-Pro-Asn-Pro confirmed that its physiological function was as a repressor of activity. More importantly, our results proved that the propeptide inhibited the activity of trypsinogen after its successful folding.