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Query: UMLS:C1389183 (autodigestion)
 317 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Intrapancreatic activation of proteases is believed to play a major role in the pathogenesis of acute necrotizing pancreatitis. Several authors have questioned, however, the central role of trypsin in autodigestion of the pancreas. To clarify the direct effects of pancreatic enzymes and other related factors on acinar cells, we used the model of isolated pancreatic acini. Acini were prepared from male Wistar rats by collagenase digestion. Protein synthesis was measured by incubation of acini with [35S]methionine. Acini were resuspended thereafter in fresh buffer and further incubated for 30-90 min under various conditions [e.g., with pancreatic homogenates, ascites (from rats with pancreatitis induced by sodium taurocholate), pure pancreatic enzymes, and other factors]. The percentage of release of newly synthesized proteins into the culture medium was regarded as a biochemical parameter of cellular integrity. A morphologic score of cellular integrity was obtained via light microscopic evaluation of acini at the end of the various incubations by measuring the degree of cell lysis, loss of cell granules, ballooning, formation of vacuoles, and karyopyknosis. When normal [35S]methionine-labeled pancreatic acini were incubated with various factors, the percentage of release of labeled proteins into the medium was as follows: incubation with HEPES/Ringer's buffer, 1.8%; hemorrhagic pancreatic ascites, 3.8%; pancreatic homogenates, 2.0%; lipase, 1.8%; phospholipase A2, 3.0%; phospholipase A2 + lecithin, 3.2%; trypsin, 2.5%; 5% olive oil, 1.8%; ascites + olive oil, 78.3%; ascites + homogenized epididymal fat, 79.9%; lipase + olive oil, 32.0%; pancreatic homogenates + olive oil, 28.0%; diolein, 2.65%; and oleic acid, 62.9%. The cellular release of radiolabeled proteins showed an inverse correlation with cellular integrity as shown by light microscopy. We postulate that interstitial release of degradation products from triglycerides by lipase causes cellular disruption. Whereas phospholipase A2 and proteases do not seem to be very harmful in the early phases of cellular damage, lipase may play a major role in acute necrotizing pancreatitis.
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PMID:Role of pancreatic enzymes and their substrates in autodigestion of the pancreas. In vitro studies with isolated rat pancreatic acini. 291 45

The specific susceptibility of the intestinal mucosa to low blood flow states is related to the "physiologic" makeup of the intestinal milieu. Pancreatic proteases appear to play a crucial role in the ischemic autodigestion of the intestinal mucosa. Moreover, trypsin can activate the conversion of xanthine dehydrogenase into superoxide radicals producing xanthine oxidase. Oxygen-derived free radicals account for at least part of the damage to the postischemic intestinal mucosa.
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PMID:Pancreatic proteases and oxygen-derived free radicals in acute ischemic enteropathy. 351 Apr 80

The effects of biliary diversion on pancreatic enzyme activities of intestinal contents was studied in conscious rats prepared with biliary and pancreatic fistulae. Diversion of bile from the intestine for 1 day caused on 80% decrease in trypsin and chymotrypsin activities of intestinal contents, in spite of increased (230%) pancreatic trypsin and chymotrypsin secretion. Bile diversion in fed rats caused a smaller decrease (58%) in trypsin and chymotrypsin activities of intestinal contents. Sodium taurocholate (100 mumol/hr intraduodenally) partially reversed the changes in pancreatic secretion and intestinal contents' activities of trypsin and chymotrypsin caused by bile diversion. The results indicated that bile was important in controlling the rate of disappearance of trypsin and chymotrypsin activities from the small intestine. The mechanism for this was studied by comparing the rate of disappearance of trypsin activity in vivo and in vitro. Bovine trypsin, with or without sodium taurocholate, was infused intraduodenally into conscious rats deprived of bile-pancreatic juice and the recovery of trypsin activity from the small intestine determined. Taurocholate increased recovery of trypsin from the small intestine more than threefold, but inactivation of bovine trypsin in vitro was not retarded by sodium taurocholate. The results indicate that bile in the small intestine controls the rate of disappearance of intraluminal trypsin and chymotrypsin activities, probably by inhibiting their autodigestion in vivo. We previously reported that bile duct ligation in rats caused decreased trypsin and chymotrypsin activities in the small intestine, but increased pancreatic enzyme secretion. We concluded that trypsin and chymotrypsin underwent accelerated inactivation in the small intestine in the absence of bile. The present study was designed to explore the mechanism for the effects of bile deprivation on intraluminal proteolytic enzyme activities in the rat.
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PMID:Importance of bile in regulation of intraluminal proteolytic enzyme activities in the rat. 615 98

There was no significant difference between the rates of protein degradation in cells of a leupeptin-producing strain and that of a leupeptin-nonproducing strain, the latter being derived from the former by mutation. Protein autodigestion in a cell homogenate of the leupeptin producer was sensitive to EDTA and chymotrypsin and less sensitive to leupeptin. On the contrary, protein degradation caused by exogenous trypsin in a similar homogenate was highly sensitive to leupeptin. A labeling experiment with [14C]-arginine of a culture of the leupeptin producer strain revealed that leupeptin was accumulated mostly in the medium and only slightly in the cells; the ratio between the amount in the medium and that in the cells was about 250:1. In contrast, leupeptin acid, the proximal intermediate having no antiplasmin activity, showed a ratio of 5:1.
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PMID:Biosynthesis of leupeptin. IV. Is protein turnover in leupeptin producer cells affected by leupeptin? 745 69

Study of the isoenzymatic spectrum of nuclear DNase from rat liver demonstrated that the extracts obtained in the presence of the protease inhibitor, PMSF, contained four polypeptides with molecular masses of 120, 54, 31 and 28 kDa possessing the endo-DNase activity. Long-term storage at -20 degrees C and autodigestion of the nuclei revealed the presence of additional polypeptides with a lower molecular mass and possessing an endo-DNase activity. Treatment of rat liver nuclear extracts with trypsin caused the appearance of an active polypeptide (M(r) 145 kDa). This finding and the multiplicity of endo-DNases of different molecular masses suggest the existence of a precursor possessing a much higher molecular mass. Chromatographic separation of endo-DNases on Sephacryl S-300 revealed three fractions of 400 and more kDa possessing a Ca2+/Mg(2+)-dependent activity, however, only after trypsin treatment.
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PMID:[Characteristics of nuclear endo-DNAase from rat liver by molecular mass and cationic dependence]. 766 4

A new proteolytic assay is described involving Coomassie blue. Under specified conditions, the amount of Coomassie-stained casein protein hydrolyzed by several proteases was proportional to the amount of protease. Coomassie dye reaction was used directly to determine the change in protein concentration of the substrate casein during proteolysis by three proteases: stem bromelain, papain, and trypsin. This method can be used with 0.1- to 0.5-micrograms quantities of protease. The dye reagent was used directly on the protease protein in order to obtain an assay of autodigestion. Autodigestion of bromelain at 50 and 25 degrees C was followed by measuring the amount of residual protease protein with time.
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PMID:Protease activity and autodigestion (autolysis) assays using Coomassie blue dye binding. 848 11

This study compares the susceptibility of pancreatic acinar cells and zymogen granules against oxidative injury and analyzes the mechanisms involved. Zymogen granules and acinar cells, isolated from rat pancreas, were exposed to a reaction mixture containing xanthine oxidase, hypoxanthine, and chelated iron. Cell function and viability were assessed by various techniques. Trypsin activation was quantified by an Elisa for trypsinogen activating peptide. Integrity of granules was determined by release of amylase. The reaction mixture rapidly generated radicals as assessed by deoxyribose and luminol assays. This oxidative stress caused lysis of granules in a matter of minutes but significant cell death only after some hours. Nevertheless, radicals initiated intracellular vacuolization, morphological damage to zymogen granules and mitochondria, increase in trypsinogen activating peptide, and decrease in ATP already after 5-30 min. Supramaximal caerulein concentrations also caused rapid trypsin activation. Addition of cells but not of granules reduced deoxyribose oxidation, suggesting that intact cells act as scavengers. Caerulein pretreatment only slightly increased the susceptibility of cells but markedly that of granules. In conclusion, isolated zymogen granules are markedly more susceptible to oxidative injury than intact acinar cells, in particular, in early stages of caerulein pancreatitis. The results show that oxidative stress causes a rapid trypsin activation that may contribute to cell damage by triggering autodigestion. Zymogen granules and mitochondria appear to be important targets of oxidative damage inside acinar cells. The series of intracellular events initiated by oxidative stress was similar to changes seen in early stages of pancreatitis.
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PMID:Oxidative injury to isolated rat pancreatic acinar cells vs. isolated zymogen granules. 874 74

Hereditary pancreatitis (HP) is a rare, early-onset genetic disorder characterized by epigastric pain and often more serious complications. We now report that an Arg-His substitution at residue 117 of the cationic trypsinogen gene is associated with the HP phenotype. This mutation was observed in all HP affected individuals and obligate carriers from five kindreds, but not in individuals who married into the families nor in 140 unrelated individuals. X-ray crystal structure analysis, molecular modelling, and protein digest data indicate that the Arg 117 residue is a trypsin-sensitive site. Cleavage at this site is probably part of a fail-safe mechanism by which trypsin, which is activated within the pancreas, may be inactivated; loss of this cleavage site would permit autodigestion resulting in pancreatitis.
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PMID:Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. 884 Nov 72

We report a rapid method for identifying proteins resolved by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) using matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). In-gel digestion was performed in a way such that the volume ratio of trypsin solution to gel plug was quantitatively controlled to promote reproducible digestion and to maximize the digestion yield. To make the digestion samples more compatible with MALDI-MS, the volatile salt ammonium bicarbonate in the digestion buffer was largely removed prior to peptide extraction. Samples of mixed tryptic peptides from in-gel digestion were used without purification to obtain molecular weights by MALDI-MS with alpha-cyano, 4-hydroxy-cinnamic acid as the matrix. Modifications of MALDI sample loading procedures improved the detection sensitivity by one half to one order of magnitude. The peptide mass peaks in MALDI-MS spectra were distinguished from those of impurities by using several types of controls, and masses were corrected by using trypsin autodigestion fragments as internal calibration standards. Two different peptide-matching computer programs were used to interrogate sequence databases and identify proteins. Identification was enhanced by generation of orthogonal data sets (by using different proteases) and by including experimental values of isoelectric point (pI) and molecular weight to exclude false entries in the candidate lists. Approximately 1% of the material from a spot was used in each sample loading, and nine protein spots from rat liver 2-D PAGE gels were identified correctly, as judged by comparison with identification results previously obtained from Edman sequencing. A previously identified low-abundance spot was not identified by MALDI-MS, presumably because there was insufficient material in a single gel. The sample handling procedure reported here should permit us to identify many 2-D PAGE protein spots of medium abundance.
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PMID:Rapid mass spectrometric identification of proteins from two-dimensional polyacrylamide gels after in gel proteolytic digestion. 915 Sep 17

Due to autodigestion the activity of dissolved trypsin successively decreases. Autolysis leads to proteolytic cleavages of some arginyl and lysyl peptide bonds of the trypsin structure. Three important autolysis sites have been reported for bovine trypsin: Lys61-Ser62, Arg117-Val118 and Lys145-Ser146. Out of these three sites only the first two exist in rat trypsin, an enzyme that has been the target of protein engineering for more than ten years. In this work Lys61 and Arg117 were replaced by Asn via site directed mutagenesis to transform the corresponding peptide bonds to trypsin resistant ones. Kinetic parameters of K61N, R117N and the double mutant K61N/R117N are practically identical with those of the wild-type enzyme. By contrast, the rate of autolysis of each singly-substituted species is substantially slower than with the parent trypsin. In particular, the double mutant shows dramatically increased stability against autolysis and decreased sensitivity to Ca2+. The process of autolysis has been followed by N-terminal sequence determination. We propose a model to explain why these two positions play a key role in autolysis and how Ca2+ can influence this process. In addition, our in vitro results strongly support the recently proposed model of human hereditary pancreatitis.
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PMID:Two mutations in rat trypsin confer resistance against autolysis. 947 79


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