Digestive end products release pancreatic enzymes from particulate cellular pools, particularly zymogen granules

The effects of various amino acids and phosphorylated forms of glucose on the release of digestive enzymes from particulate cellular pools, particularly zymogen granules, were evaluated in rat pancreas. Whole tissue homogenates, as well as zymogen granules isolated either by differential centrifugation in 0.3 M sucrose or by preparation in buffered sucrose and subsequent centrifugation in a Percoll gradient, were studied. The basic amino acids L-arginine and L-lysine, sites of tryptic cleavage, caused the release of trypsinogen, but not chymotrypsinogen, whereas the aromatic amino acids L-phenylalanine and L-tryptophan, sites of chymotryptic cleavage, caused release of both trypsinogen and chymotrypsinogen. Neither led to the release of the starch-splitting enzyme amylase. All effects occurred within the range of normal plasma concentrations for these amino acids in the rat. Two amino acids, L-threonine and hydroxy-L-proline, that are not sites of cleavage by trypsin or chymotrypsin, and a nonmammalian amino acid, aminoadipic acid, did not lead to release of trypsinogen, chymotrypsinogen, or amylase. Two phosphorylated forms of glucose, glucose 1-phosphate and glucose 1,6-diphosphate, caused the release of amylase, but of neither trypsinogen nor chymotrypsinogen. Contrary to previous results, D-glucose was without effect, as was glucose 6-phosphate. We propose that certain digestive end products, by direct action on zymogen granules, cause the selective release of the enzymes involved in their evolution from polymeric substrates during digestion.     

Absence of the major zymogen granule membrane protein, GP2, does not affect pancreatic morphology or secretion

The majority of digestive enzymes in humans are produced in the pancreas where they are stored in zymogen granules before secretion into the intestine. GP2 is the major membrane protein present in zymogen granules of the exocrine pancreas. Numerous studies have shown that GP2 binds digestive enzymes such as amylase, thereby supporting a role in protein sorting to the zymogen granule. Other studies have suggested that GP2 is important in the formation of zymogen granules. A knock-out mouse was generated for GP2 to study the impact of the protein on pancreatic function. GP2-deficient mice displayed no gross signs of nutrient malab-sorption such as weight loss, growth retardation, or diarrhea. Zymogen granules in the GP2 knock-out mice appeared normal on electron microscopy and contained the normal complement of proteins excluding GP2. Primary cultures of pancreatic acini appropriately responded to secretagogue stimulation with the secretion of digestive enzymes. The course of experimentally induced pancreatitis was also examined in the knock-out mice because proteins known to associate with GP2 have been found to possess a protective role. When GP2 knock-out mice were subjected to two different models of pancreatitis, no major differences were detected. In conclusion, GP2 is not essential for pancreatic exocrine secretion or zymogen granule formation. It is unlikely that GP2 serves a major intracellular role within the pancreatic acinar cell and may be functionally active after it is secreted from the pancreas.     

Feeding activates protein synthesis in mouse pancreas at the translational level without increase in mRNA

To determine the mechanism of meal-regulated synthesis of pancreatic digestive enzymes, we studied the effect of fasting and refeeding on pancreatic protein synthesis, relative mRNA levels of digestive enzymes, and activation of the translational machinery. With the use of the flooding dose technique with L-[3H]phenylalanine, morning protein synthesis in the pancreas of Institute for Cancer Research mice fed ad libitum was 7.9 +/- 0.3 nmol phenylalanine.10 min(-1).mg protein(-1). Prior fasting for 18 h reduced total protein synthesis to 70 +/- 1.4% of this value. Refeeding for 2 h, during which the mice consumed 29% of their daily food intake, increased protein synthesis to 117.3 +/- 4.9% of the control level. Pancreatic mRNA levels of amylase, lipases, trypsins, chymotrypsin, elastases, as well as those for several housekeeping genes tested were not significantly changed after refeeding compared with fasted mice. By contrast, the major translational control pathway involving Akt, mTOR, and S6K was strongly regulated by fasting and refeeding. Fasting for 18 h decreased phosphorylation of ribosomal protein S6 to almost undetectable levels, and refeeding highly increased it. The most highly phosphorylated form of the eIF4E binding protein (4E-BP1) made up the 14.6% of total 4E-BP1 in normally fed animals, was only 2.8% after fasting, and was increased to 21.4% after refeeding. This was correlated with an increase in the formation of the eIF4E-eIF4G complex after refeeding. By contrast, feeding did not affect eIF2B activity. Thus food intake stimulates pancreatic protein synthesis and translational effectors without increasing digestive enzyme mRNA levels.     

Maturation of the intestinal digestion and of microbial activity in the young rabbit: impact of the dietary fibre:starch ratio.

The developmental changes of intestinal digestive potential and caecal microbial activity were described in suckling and weaned rabbits according to two feeding programmes. Two groups of thirteen litters were fed from 18 to 42 days old a "High" or a "Medium" NDF:starch ratio diet (resp. 2.7 vs 2.0, groups HL and ML) with similar protein and lipid levels, and from 42 to 70 days old the two groups were fed a "Low" NDF:starch ratio diet (1.7). From 25 to 32 days (weaning), the milk and solid feed intake were 22% and 41% higher in ML group (P<0.05), and the mortality by diarrhoea was 4 units lower (P<0.01). The whole tract digestive efficiency increased by 10% before weaning, and remained steady (organic matter) or decreased (lipids, protein) after weaning. Energy digestibility was 0.623 and 0.686 for High and Medium diets respectively. From 25 to 42 days, total enzymatic activity in intestinal content increased for chymotrypsin (5-fold, P<0.001), lipase (10-fold, P<0.001), amylase (17-fold, P<0.01) and maltase (11-fold, P<0.001), while trypsin doubled after weaning. The feeding programme only affected the amylase and maltase activities, that were higher in HL group (P<0.05). The volatile fatty acids concentration in the caecum was not significantly different among the groups, but it increased by 44% 10 days after weaning. The bacterial fibrolytic enzymes, increased by 30% after weaning and were similar among the two groups. The study revealed that the intestinal digestive maturation and the caecal microbial activity of the rabbit evolved markedly between 3 and 5 weeks of age, and was weakly affected when the NDF:starch ratio decreased from 2.7 to 2.0.     

Development of pancreatic acini in embryos of the grass snake Natrix natrix (Lepidosauria,Serpentes)

This study report about the differentiation of pancreatic acinar tissue in grass snake, Natrix natrix, embryos using light microscopy, transmission electron microscopy, and immuno-gold labeling. Differentiation of acinar cells in the embryonic pancreas of the grass snake is similar to that of other amniotes. Pancreatic acini occurred for the first time at Stage VIII, which is the midpoint of embryonic development. Two pattern of acinar cell differentiation were observed. The first involved formation of zymogen granules followed by cell migration from ducts. In the second, one zymogen granule was formed at the end of acinar cell differentiation. During embryonic development in the pancreatic acini of N. natrix, five types of zymogen granules were established, which correlated with the degree of their maturation and condensation. Within differentiating acini of the studied species, three types of cells were present: acinar, centroacinar, and endocrine cells. The origin of acinar cells as well as centroacinar cells in the pancreas of the studied species was the pancreatic ducts, which is similar as in other vertebrates. In the differentiating pancreatic acini of N. natrix, intermediate cells were not present. It may be related to the lack of transdifferentiation activity of acinar cells in the studied species. Amylase activity of exocrine pancreas was detected only at the end of embryonic development, which may be related to animal feeding after hatching from external sources that are rich in carbohydrates and presence of digestive enzymes in the egg yolk. Mitotic division of acinar cells was the main mechanism of expansion of acinar tissue during pancreas differentiation in the grass snake embryos.     

Evidence that amylase is released from two distinct pools of secretory proteins in the pancreas

Previous experiments demonstrated the existence of at least two pools of secretory proteins in the exocrine pancreas. We have measured the specific activities of amylase released under resting conditions and of amylase in the zymogen granules. Specific activity of resting secretion was twice that found under stimulated conditions or in zymogen granules. Secretory proteins were pulse-labeled and amylase was measured after precipitation of the enzyme with glycogen. Pancreatic juice collected at 45-50 min post-pulse contained 10-25-times the amylase activity found in zymogen granules. These results confirm the existence of at least two distinct pools of secretory proteins in the exocrine pancreas and suggest the existence of an intracellular route of secretory proteins which would bypass the zymogen granule compartment.     

Postprandial responses in the African rhombig egg eater (Dasypeltis scabra)

The African rhombic egg eater (Dasypeltis scabra) is a colubrid snake feeding exclusively on bird eggs. Frequency of feeding is governed by the seasonal availability of bird eggs; i.e., long fasting intervals change with relatively short periods when plenty of food is available. Intermittent feeding snakes show a remarkable postprandial increase of metabolic rate and digestive organ size. The postprandial increase in metabolic rate (specific dynamic action, SDA) in snakes is affected by meal size, temperature, and meal composition. A major portion of SDA in snakes is allocated to gastric function and the breakdown of the meal. We hypothesize that SDA in egg eaters is lower than in other snake species, because egg eaters feed on “liquid” food that does not require enzymatic breakdown in the stomach. We also hypothesized that other components of the postprandial response of egg eaters (e.g., size changes of the intestine and the liver) do not differ from other snakes. The standard metabolic rate and metabolic response to feeding were measured using closed-chamber respirometry. Size changes of small intestine and liver were measured using high-resolution transcutaneous ultrasonography. Standard metabolic rates of fasting egg eaters were in the same range of mass specific values as known from other snakes. Within 24 h after feeding, oxygen consumption doubled and peaked at 2 days after feeding. At the same time, the size of the small intestine and the cross-sectional diameter of the liver increased. Within 2 days after feeding, the size of the mucosal epithelium doubled its thickness. Liver size increased significantly within 24 h reaching maximum size 2–4 days after feeding. The size of both organs returned to fasting values within 7–10 days after feeding. The postprandial response of African rhombic egg eaters shows the same pattern and dynamics as known from other snake species. However, the factorial increase of metabolic rate during SDA is the lowest reported for any snake. A comparison with literature data supports the idea that SDA is mainly determined by gastric function and that it is low in egg eaters because they do not have to break down solid meals in the stomach as other snake species do.     

Responses of the exocrine pancreatic secretion to spontaneous feeding in rats with bile-pancreatic juice diversion

The regulatory response of the exocrine pancreas was examined in rats under unanesthetized and unrestrained conditions. The previous study demonstrated that the pancreatic protease secretion increased 2-fold after spontaneous feeding of a low protein diet in chronically bile-pancreatic cannulated rats (normal rats) whose bile-pancreatic juice (BPJ) was returned to the duodenum. In the present study, we observed the response of the exocrine pancreatic secretion to spontaneous feeding of a low protein diet in rats with chronic diversion of BPJ from the proximal small intestine for 6 days (bypass rat) whose diverted BPJ was returned to the upper ileum. During BPJ diversion, the dry weight and the protein content of the pancreas were increased 2-fold, compared with normal rats. Also, the levels of trypsinogen and chymotrypsinogen in the pancreas were increased several times, but amylase was decreased. The basal secretion of enzymes after a 24-hr fast was enhanced in bypass rats in proportion to the pancreatic enzyme contents. After spontaneous feeding of 8% casein fat-free diet, the increases in the pancreatic secretion of bypass rats were much smaller than those of normal rats. In contrast, the increase of BPJ flow of bypass rats after feeding was greater than that of normal rats. These findings represent that the chronic diversion of BPJ exerts hypergrowth of pancreas and hypersecretion of proteases in the fasting state, and less sensitivity of pancreatic enzyme secretion to dietary feeding.     

Effect of a high-protein diet on the gene expression of a trypsin-sensitive, cholecystokinin-releasing peptide (monitor peptide) in the pancreas

The adaptation to a high protein diet of the concentration and mRNA level of a trypsin-sensitive, cholecystokinin-releasing peptide (monitor peptide), which was proposed to be the mediator of the cholecystokinin release in response to protein intake, was investigated in the rat pancreas. Adult rats were placed on one of two isocaloric diets. One group was fed a 22% casein diet (control diet) and the other a 64% casein diet (high-protein diet) for 14 days. In order to quantify the monitor peptide separately from pancreatic secretory trypsin inhibitor (PSTI-II), which is highly similar in its amino acid and mRNA nucleotide sequences to the monitor peptide but has less cholecystokinin-releasing activity, we used specific assay methods: HPLC was used for determining the monitor peptide concentration in zymogen granules and a synthetic oligonucleotide probe for determining the mRNA of the monitor peptide in the pancreas. The concentrations in the zymogen granules and the mRNA levels in the pancreas of the two peptides increased in parallel during the adaptation to the high protein diet, indicating that these two peptides were under the same control during the adaptation. The concentration and mRNA level of the monitor peptide, which were measured after 0, 3, and 14 days, increased throughout the experiment period, as did the concentration of trypsin. This suggested that the monitor peptide and trypsin may respond to similar signals during the adaptation to a high protein diet and that this apparent coordination may facilitate the adaptation of the pancreas to the diet.     

Developmental changes in digestive physiology of nestling house sparrows, Passer domesticus

Six decades of studies have speculated that digestive capacity might limit avian growth rate or that developmental changes in the gut might determine developmental changes in digestive efficiency. However, there are no studies on digestive enzymes during avian development, except for studies on mainly domestic birds that exhibit the precocial mode of development. We studied alimentary organ masses, intestinal enzyme activities (sucrase, maltase, isomaltase, aminopeptidase-N), and pancreatic enzyme activities (amylase, trypsin, chymotrypsin) during development of a wild passerine bird exhibiting the altricial mode of development. Wild nestling house sparrows were studied immediately after removal from the nest (days 0, 3, 6 of age; day 0=hatch), whereas captives were raised in the laboratory beginning day 3 on a formulated casein/starch-based diet until fledging age (after day 12). Digestive biochemistry was dynamic. Tissue-specific activities of some digestive enzymes continued to increase through fledging, by >10 times in some cases (e.g., sucrase and maltase in midintestine). Total pancreatic amylase activity increased 100 times between hatch and day 12 through a combination of increases in tissue-specific activity and pancreas mass. House sparrows differ from poultry, in whom after about 2 wk of age the specific activity of intestinal and pancreatic digestive enzymes is generally constant or declines during development. The data on intestinal and pancreatic enzymes help explain why digestive efficiency of nestling house sparrows improves with age, and the data seem consistent with the idea that digestive capacity might limit feeding rate and hence growth rate.     

Luminal dietary protein, not amino acids, induces pancreatic protease via CCK in pancreaticobiliary-diverted rats

We determined whether pancreatic adaptation to a high-protein diet depends on ingested protein in the intestinal lumen and whether such adaptation depends on a CCK or capsaicin-sensitive vagal afferent pathway in pancreaticobiliary-diverted (PBD) rats. Feeding a high-casein (60%) diet but not a high-amino acid diet to PBD rats increased pancreatic trypsin and chymotrypsin activities compared with those after feeding a 25% casein diet. In contrast, feeding both the high-nitrogen diets induced pancreatic hypertrophy in PBD rats. These pancreatic changes by the diets were abolished by treatment with devazepide, a CCK-A receptor antagonist. Protease zymogen mRNA abundance in the PBD rat was not increased by feeding the high-casein diet and was decreased by devazepide. Perivagal capsaicin treatment did not influence the values of any pancreatic variables in PBD rats fed the normal or high-casein diet. We concluded that luminal protein or peptides were responsible for the bile pancreatic juice-independent induction of pancreatic proteases on feeding a high-protein diet. The induction was found to be dependent on the direct action of CCK on the pancreas. Pancreatic growth induced by high-protein feeding in PBD rats may depend at least partly on absorbed amino acids.     

Chymotrypsin C is a co-activator of human pancreatic procarboxypeptidases A1 and A2

Human digestive carboxypeptidases CPA1, CPA2, and CPB1 are secreted by the pancreas as inactive proenzymes containing a 94-96-amino acid-long propeptide. Activation of procarboxypeptidases is initiated by proteolytic cleavage at the C-terminal end of the propeptide by trypsin. Here, we demonstrate that subsequent cleavage of the propeptide by chymotrypsin C (CTRC) induces a nearly 10-fold increase in the activity of trypsin-activated CPA1 and CPA2, whereas CPB1 activity is unaffected. Other human pancreatic proteases such as chymotrypsin B1, chymotrypsin B2, chymotrypsin-like enzyme-1, elastase 2A, elastase 3A, or elastase 3B are inactive or markedly less effective at promoting procarboxypeptidase activation. On the basis of these observations, we propose that CTRC is a physiological co-activator of proCPA1 and proCPA2. Furthermore, the results confirm and extend the notion that CTRC is a key regulator of digestive zymogen activation.     

Effects of GP2 expression on secretion and endocytosis in pancreatic AR4-2J cells

GP2 is the major membrane protein present in secretory granules of the exocrine pancreas. GP2's function is unknown, but a role in digestive enzyme packaging or secretion from secretory granules has been proposed. In addition, GP2 has been proposed to influence endocytosis and membrane recycling following stimulated secretion. Adenovirus-mediated GP2 overexpression in the rat pancreatic cell line AR4-2J was used to study its impact on digestive enzyme secretion and membrane recycling. Immunoelectron microscopy showed that GP2 and amylase co-localized in secretory granules in infected AR4-2J cells. CCK-8 stimulation resulted in a fourfold increase in amylase secretion with or without GP2 expression. GP2 expression also did not influence endocytosis following CCK-8 stimulation. Thus, GP2 expression in AR4-2J cells does not affect amylase packaging in secretory granules or stimulated secretion. GP2 expression also does not influence membrane recycling in response to stimulated stimulation in AR4-2J cells.     

Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion

The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1). Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase) were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.     

Isolation of zymogen granules from rat pancreas and characterization of their membrane proteins

A zymogen granule fraction has been isolated from rat pancreas, and its purity has been assessed by biochemical and morphological criteria. Specific activities of two marker enzymes, amylase and chymotrypsin, are increased by 4.6 and 5.4-fold, respectively, as compared to the homogenate. The purified fraction is devoid of detectable RNA, DNA and 5'-nucleotidase, glucose-6-phosphatase, and cytochrome c oxidase activities. Electron micrographs confirm the absence of mitochondria, lysosomes, and rough endoplasmic reticulum fragments. Zymogen granule membranes were isolated from this fraction on a sucrose gradient following lysis in alkaline buffer. Secretory contaminants were efficiently removed from the membranes as indicated by experiments in which labeled secretory proteins were added during the isolation procedure and secondly by measuring residual levels of amylase and chymotrypsin. Three enzyme activities were found in the membranes: thiamine pyrophosphatase, ATP-diphosphohydrolase, and low levels of acid phosphatase. Membrane proteins were solubilized by urea-Triton X-100 and separated in double-dimension (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Isoelectric point and molecular weight of each protein band were determined.     

Zinc Oxide–Montmorillonite Hybrid Influences Diarrhea, Intestinal Mucosal Integrity, and Digestive Enzyme Activity in Weaned Pigs

One hundred-eighty piglets (Duroc × Landrace × Yorkshire), with an average initial weight of 7.4?kg weaned at 27?±?1?days of age, were used to evaluate the effects of dietary zinc oxide?Cmontmorillonite hybrid (ZnO?CMMT) on growth performance, diarrhea, intestinal mucosal integrity, and digestive enzyme activity. All pigs were allotted to five treatments and fed with the basal diets supplemented with 0, 250, 500, and 750?mg/kg of Zn as ZnO?CMMT or 2,000?mg/kg of Zn as ZnO. The results showed that supplementation with 500 or 750?mg/kg of Zn from ZnO?CMMT and 2,000?mg/kg of Zn from ZnO improved average daily gain, enhanced average daily feed intake, decreased fecal scores at 4, 8, and 14?days postweaning, reduced intestinal permeability which was evident from the reduced lactulose recovery and urinary lactulose/mannitol ratio, and improved the activities of protease, amylase, lipase, trypsin, and chymotrypsin both in pancreas and small intestinal contents of pigs as compared with the control. Supplemental 250?mg/kg of Zn from ZnO?CMMT also decreased fecal scores at 8 and 14?days postweaning, decreased urinary lactulose/mannitol ratio, and improved chymotrypsin activity in pancreas and small intestinal contents as well as protease activity in small intestinal contents compared with control. Moreover, the above indexes of weanling pigs fed with 500 or 750?mg/kg of Zn as ZnO?CMMT did not differ from those fed with 2,000?mg/kg of Zn as ZnO. The results demonstrated that supplementation with 500 or 750?mg/kg of Zn from ZnO?CMMT was as efficacious as 2,000?mg/kg of Zn from ZnO in improving growth performance, alleviating postweaning diarrhea, and enhancing intestinal mucosal integrity and the digestive enzyme activities in pancreas and small intestinal contents of pigs. The results that feeding lower concentrations of ZnO?CMMT to weanling pigs maintained performance will be beneficial for the environment and for sustaining swine production.     

Pancreatic adaptation to dietary lipids is mediated by changes in lipase mRNA

Lipase activity, rates of biosynthesis of lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) and amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) as well as concentrations of their corresponding mRNAs were measured in the pancreatic tissue of rats fed isocaloric and isoprotein diets with inverse changes in the amounts of lipids and carbohydrates. A control diet (3% sunflower oil--62% starch) and three lipid-rich diets (10% sunflower oil--46.2% starch, 25% sunflower oil--12.5% starch and 30% sunflower oil--1.25% starch) were fed to rats for 10 days. Ingestion of the 10% lipid diet already resulted in a 1.4-fold increase in lipase activity while a 2.4-fold increase was observed with the other 2 high-lipid low-carbohydrate diets. Similarly, 1.3- and 3.1-fold increases in the total rate of protein synthesis were measured in pancreatic lobules of rats fed 10 and 25% or 30% lipid diets, respectively, as compared with control animals. While absolute lipase synthesis showed an important increase during the dietary manipulation (1.7- and 5.9-fold, respectively), amylase synthesis was significantly lower (1.1- and 1.5-fold, respectively). The level of lipase mRNA, as measured by dot-blot hybridization with the corresponding specific cDNA, showed a 2.2-fold increase (10% lipid diet) and a 3.9-fold increase (25% lipid diet), whereas the level of amylase mRNA showed only 1.1- and 1.3-fold increases under the same experimental conditions. These data demonstrated that protein-specific synthesis rates more accurately reflected pancreatic adaptive states than tissue levels of enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of pancreatic enzyme secretion-stimulating activity and trypsin inhibitory activity in zymogen granule of the rat pancreas

The intracellular localization of pancreatic enzyme secretion-stimulating activity in rat pancreas was investigated. We found and purified a pancreatic enzyme secretion-stimulating peptide from rat bile/pancreatic juice. The peptide is trypsin-sensitive (showing temporary trypsin inhibitory activity), and it is hypothesized that it acts as a trypsin-sensitive mediator in the feedback regulation of diet-induced pancreatic enzyme secretion. The zymogen granule fraction was purified 5-fold by ultracentrifugation by the Percoll density gradient method. The purity of the zymogen granule fraction was determined from the specific amylase activity and electron microscopic morphology. The specific enzyme activities of amylase and trypsin and the trypsin inhibitory activity increased in parallel during the purification, and the pancreatic enzyme secretion-stimulating activity was also localized in the zymogen granule fraction. These results suggest that the pancreatic enzyme secretion-stimulating peptide originates from the acinar cells, and that it is secreted through exocytosis of zymogen granules into the small intestine, its ratio to trypsin thus remaining constant. This idea supports our hypothesis that the stimulating peptide acts as a mediator for the feedback regulation of pancreatic enzyme secretion by trypsin.     

Purification of chymotrypsin from bovine pancreas using precipitation with a strong anionic polyelectrolyte

The separation of chymotrypsin from a crude filtrate of bovine pancreas homogenate was carried out using precipitation with a commercially available negatively charged strong polyelectrolyte: polyvinyl sulfonate. The zymogen form of chymotrypsin was activated by addition of trypsin (0.01 mg/g homogenate), then, the enzyme was precipitated by polyelectrolyte addition at pH 2.5 in the pancreas homogenate. A stoichiometric ratio of 670 bound molecules of chymotrypsin per polyelectrolyte molecule was found in the non-soluble form of the enzyme–polyelectrolyte complex. The non-soluble complex was separated by simple centrifugation and re-dissolved by a pH change to 8.0. The recovery of chymotrypsin biological activity was 61% of the initial activity in the homogenate with 4.7-fold increase in its specific activity.     

Digestive enzymes of two freshwater fishes (Limia vittata and Gambusia punctata) with different dietary preferences at three developmental stages

The variation of activity of some digestive enzymes was studied in three age groups of two freshwater endemic fishes from Cuba: Limia vittata and Gambusia punctata. Trypsin, chymotrypsin and amylase activities showed a different pattern between both species. Trypsin and chymotrypsin activity increased with the age of fishes, while amylase activity decreased. The highest activity of trypsin and chymotrypsin was registered in G. punctata while the highest amylase activity was detected in L. vittata. Zymograms revealed proteases with molecular masses similar to trypsin and chymotrypsin reported for other fish species. Amylase electrophoresis showed the presence of this enzyme; in L. vittata amylase zymograms showed two bands with molecular masses of 175 and 100 kDa and in G. punctata four bands of 175, 100, 46 and 30 kDa respectively were found. The activity of the digestive enzymes can be used as an effective indicator of the feeding habits and the development of the digestive tracts in L. vittata and G. punctata.