Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.9 (enterokinase)
 675 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The molecular forms of immunoreactive pancreatic cationic trypsin in sera of patients with acute pancreatic inflammation have been characterized using a radioimmunoassay technique that is capable of detecting trypsinogen as well as trypsin bound to alpha 1-antitrypsin. Trypsin bound to alpha 2-macroglobulin is not immunoreactive under normal assay conditions. However, alpha 2-macroglobulin-bound trypsin can be detected after gel filtration of serum on Bio-Gel A-0.5 m and acid treatment of column fractions. The average serum level of immunoreactive cationic trypsin from 20 patients with acute pancreatic inflammation was 1,590 ng/ml. An average normal value of 26 ng/ml has been obtained previously. Serum samples from 14 patients with pancreatic inflammation were chromatographed under conditions that resolve trypsinogen, alpha 1-antitrypsin-bound trypsin, and alpha 2-macroglobulin-bound trypsin. In each case, the major portion of the immunoreactive material eluted at a position corresponding to free trypsinogen, while a minor fraction of the immunoreactive material appeared to be trypsin bound to alpha 1-antitrypsin. The zymogen nature of the major peak was confirmed in one case by activation with human enteropeptidase. In 11 of 14 patients, acid treatment of the alpha 2-macroglobulin peak yielded immunoreactive trypsin.
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PMID:Molecular forms of immunoreactive pancreatic cationic trypsin in pancreatitis patient sera. 9 23

The affinity chromatography of human enterokinase using p-aminobenzamidine as the ligand [Grant, D.A.W. & Hermon-Taylor, J. (1976) Biochem. J. 155, 243-254] has been reassessed and the optimal conditions for the synthesis and operation of the derivatised gel defined. Satisfactory adsorbants were only produced using high concentrations of both CNBr and spacer-arm in the initial coupling slurry. Under these conditions it seemed likely that the majority of the ligand in a sterically favourable position to bind enterokinase was on the external surface of the bead. Trypsin binding to the adsorbant was not so critically dependent on the synthetic conditions and correlated closely with the degree of substitution. Dilution of the adsorbant with unlabelled Sepharose 4B indicated that there was more than one binding site per enterokinase molecule. The highest affinity was presumably for the active site, with adsorption supported by secondary interactions with spacer-arm or gel matrix not necessarily on the same bead. Maximal resolution was obtained by prolonged washing of the gel after loading; two populations of intestinal aminopeptidase were identified. Substitution of aniline for p-aminobenzamidine abolished specific enterokinase adsorption and improved the purification procedure by further removal of onon-specifically adsorbed contaminants.
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PMID:Optimisation of conditions for the affinity chromatography of human enterokinase on immobilised p-aminobenzamidine. Improvement of the preparative procedure by inclusion of negative affinity chromatography with glycylglycyl-aniline. 35 95

The activities of highly purified human enterokinase (enteropeptidase, EC 3.4.21.9) and bovine trypsin were tested against three synthetic substrates alpha-N-Benzoyl-L-arginine ethyl ester HCl, alpha-N-Benzoyl-DL-arginine-p-nitroanilide HCl and alpha-N-Benzoyl-DL-arginine-2-naphthylamide HCl. There was no detectable hydrolysis of these substrates by enterokinase whereas the kinetic parameters obtained for trypsin were in close agreement with those previously described by other workers. The values for Km and kcat were dependent on the Ca2+ concentration. Hydrolysis of glycine-tetra-L-aspartyl-L-lysyl-2-naphthylamide (Gly(Asp)4-Lys-Nap) by these protease was also studied. Enterokinase-catalysed hydrolysis obeyed simple steady-state kinetics and values for Km of 0.525 mM and 0.28 mM and for kcat of 21.5 s-1 and 28.3 s-1 were obtained in 0.1 mM and 10 mM Ca2+, respectively. Trypsin-catalysed hydrolysis was complex and the response to Ca2+ was sigmoidal partly due to the lability of trypsin at low Ca2+ concentrations. A sensitive specific assay for enterokinase was developed and applied to the measurement of the enzyme in serum; interference by nonspecific arylamidases was eliminated by the addition of Zn2+.
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PMID:Hydrolysis of artificial substrates by enterokinase and trypsin and the development of a sensitive specific assay for enterokinase in serum. 45 24

Enterokinase is an enzyme produced by the mucosa of the small intestine. Its sole function is to activate trypsinogen to trypsin. In animals and man the duodenum and proximal jejunum have high levels of activity whereas the remaining small bowel has minimal levels. A reproducible assay was developed for measuring mucosal enterokinase activity applicable to operative and endoscopic biopsies. Anaesthetic and operative techniques were developed for small intestinal resections in guinea-pigs to ensure their long term survival. Transposition of high-enterokinase-secreting segments of guinea-pig small intestine to low-enterokinase regions and vice versa showed no alteration of enterokinase activity in the transposed segments. Similarly, resection of the enterokinase region in five proximal pancreaticoduodenectomy operations in man revealed no induction of enterokinase in the remaining jejunum at endoscopy 6 months later. Isolation of high-enterokinase-secreting segments of small bowel from their luminal continuity by fashioning of Thiry--Vella fistulas led to a decay of enterokinase activity to minimal levels within 12--16 h. Perfusion of these fistulas with trypsin and sodium, or chymotrypsin and sodium, prevented this decay. If the enterokinase was allowed to decay over 24 h its activity could be restored to 80 per cent of its normal level by perfusion for 24 h with trypsin and sodium. Trypsin and sodium acti in combination on an enterocyte membrane receptor to stimulate enterokinase synthesis.
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PMID:Regulation of enterokinase synthesis in animal and human small intestine by luminal signals: its implication in upper gastrointestinal surgery. 50 46

The two human anionic trypsinogens 1 and 2 were purified from human pancreatic juice by gel filtration on Sephadex G-100 and by chromatography on DEAE-cellulose. After activation of their respective zymogens by porcine enterokinase, human trypsins 1 and 2 were studied for their reaction with a wide variety of proteinase inhibitors. Kunitz pancreatic trypsin inhibitor and human pancreatic secretory trypsin inhibitor completely inhibited both human trypsins at a stoichiometric inhibitor-to-enzyme ratio of one to one. In contrast, bovine pancreatic secretory trypsin inhibitor (Kazal's inhibitor) failed to inhibit either human trypsin. The inhibition of both human trypsins by porcine pancreatic secretory trypsin inhibitor was demonstrated. The reactions of the trypsins with chicken ovomucoid, Ascaris lumbricoides (type suis), human sperm and blood plasma trypsin inhibitors were studied. The most striking difference between the two human trypsins was the reaction with soybean trypsin inhibitor (Kunitz). Trypsin 2 was completely inhibited in a one-to-one molar ratio while trypsin 1 was poorly inhibited. The presence of a prekallikrein in human pancreatic juice is discussed.
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PMID:The two human trypsinogens. Inhibition spectra of the two human trypsins derived from their purified zymogens. 107 68

For the purpose of resolving the mechanism of the ill-effect of refluxed pancreatic juice, due to the anomalous pancreaticobiliary ductal union, canine models were produced. Mongrel adult dogs and puppies were used. Pancreatico-cholecystostomy was performed on 5 adult dogs and 5 puppies. Pancreatico-choledochostomy was performed on 10 adult dogs and 5 puppies. In both procedures, the cylindrical dilatation of the choledochus resulted. The pathological changes in the choledochus were very similar to the cylindrical choledochal dilatation in the human. Elastic fibers diminished universally and in some cases they vanished completely. The changes in the papilla of the bile duct were very slight. There were no cases with hardened papilla. The pathological changes were greater in the pancreatico-cholecystostomy. Enzymatic analysis of pancreatic juice in bile was very important in explaining the etiology. Amylase was proven to be very high throughout the cases, over 10,000 IU/1 by enzymatic assay. Trypsin and elastase were proven to be activated in pancreatico-cholecystostomy, naturally assuming the existence of enterokinase. They were aso proved to be activated in the pancreatico-choledochostomy, without the existence of enterokinase. Though the mechanism was not clear, the activated proteases were the actual cause of the ill-effect in this anomaly. Thus we have succeeded in producing the ideal animal models of the reflux of pancreatic juice into the bile duct in adult dogs and puppies.
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PMID:The production of anomalous pancreaticobiliary ductal union in canine models. 728 70

Histidine 57 of the catalytic triad of trypsin was replaced with alanine to determine whether the resulting variant would be capable of substrate-assisted catalysis [Carter, P., & Wells, J. A. (1987) Science 237, 394-9]. A 2.5-fold increase in kcat/Km was observed on tri- or tetrapeptide substrates containing p-nitroanilide leaving groups and histidine at P2. In contrast, hydrolysis of peptide substrates extending from P6 to P6' is improved 70-300-fold by histidine in the P2 or P1' position. This preference creates new protease specificities for sequences HR decreases, R decreases H, HK decreases, and K decreases H. The ability of histidine from either the P2 or the P1' position of substrate to participate in catalysis emphasizes the considerable variability of proteolytically active orientations which can be assumed by the catalytic triad. Trypsin H57A is able to hydrolyze fully folded ornithine decarboxylase with complete specificity at a site containing the sequence HRH. Trypsin H57A was compared to enteropeptidase in its ability to cleave a propeptide from trypsinogen. Trypsin H57A cleaved the propeptide of a variant trypsinogen containing an introduced FPVDDDHR cleavage site only 100-fold slower than enteropeptidase cleaved trypsinogen. The selective cleavage of folded proteins suggests that trypsin H57A can be used for specific peptide and protein cleavage. The extension of substrate-assisted catalysis to the chymotrypsin family of proteolytic enzymes indicates that it may be possible to apply this strategy to a wide range of serine proteases and thereby develop various unique specificities for peptide and protein hydrolysis.
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PMID:Trypsin specificity increased through substrate-assisted catalysis. 754 82

Trypsin is a highly valuable protease that has many industrial and biomedical applications. The growing demand for non-animal sources of the enzyme and for trypsins with special properties has driven the interest to clone and express this protease in microorganisms. Reports about expression of recombinant trypsins show wide differences in the degree of success and are contained mainly in patent applications, which disregard the difficulties associated with the developments. Although the yeast Pichia pastoris appears to be the microbial host with the greatest potential for the production of trypsin, it has shown problems when expressing cold-adapted fish trypsins (CAFTs). CAFTs are considered of immense value for their comparative advantage over other trypsins in a number of food-processing and biotechnological applications. Thus, to investigate potential obstacles related to the production of CAFTs in P. pastoris, the cunner fish trypsin (CFT) was cloned in different Pichia expression vectors. The vectors were constructed targeting both internal and secreted expression and keeping the CFT native signal peptide. Western-blotting analysis confirmed the expression with evident differences for each construct, observing a major effect of the leader peptide sequence on the expression patterns. Immobilized nickel affinity chromatography yielded a partially purified recombinant CFT, which exhibited trypsin-specific activity after activation with bovine enterokinase.
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PMID:Expression of a cold-adapted fish trypsin in Pichia pastoris. 1592 13

Enteropeptidase, a type II transmembrane serine protease, is localized to the brush border of the duodenal and jejunal mucosa. It is synthesized as a zymogen (proenteropeptidase) that requires activation by another protease, either trypsin or possibly duodenase. Active enteropeptidase then converts the pancreatic precursor, trypsinogen, to trypsin by cleavage of the specific trypsinogen activation peptide, Asp-Asp-Asp-Asp-Lys- Ile that is highly conserved in vertebrates. Trypsin, in turn, activates other digestive zymogens such as chymotrypsinogen, proelastase, procarboxypeptidase and prolipase in the lumen of the gut. The important biological function of enteropeptidase is highlighted by the manifestation of severe diarrhea, failure to thrive, hypoproteinemia and edema as a result of congenital deficiency of enteropeptidase activity in the gut. Conversely, duodenopancreatic reflux of proteolytically active enteropeptidase may cause acute and chronic pancreatitis.
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PMID:Enteropeptidase, a type II transmembrane serine protease. 1948 41

1. A powerful kinase which changes trypsinogen to trypsin was found to be present in the synthetic liquid culture medium of a mold of the genus Penicillium. 2. The concentration of kinase in the medium is increased gradually during the growth of the mold organism and continues to increase for some time even after the mold has ceased growing. 3. Mold kinase transforms trypsinogen to trypsin only in an acid medium. It differs thus from enterokinase and trypsin which activate trypsinogen best in a slightly alkaline medium. 4. The action of the mold kinase in the process of transformation of trypsinogen is that of a typical enzyme. The process follows the course of a catalytic unimolecular reaction, the rate of formation of a definite amount of trypsin being proportional to the concentration of kinase added. The ultimate amount of trypsin formed, however, is independent of the concentration of kinase used. 5. The formation of trypsin from trypsinogen by mold kinase is not accompanied by any measurable loss of protein. 6. The temperature coefficient of formation of trypsin from trypsinogen by mold kinase varies from Q(5-15) = 1.70 to Q(25-30) = 1.25 with a corresponding variation in the value of micro from 8100 to 4250. 7. Trypsin formed from trypsinogen by means of mold kinase is identical in crystalline form with the crystalline trypsin obtained by spontaneous autocatalytic activation of trypsinogen at pH 8.0. The two products have within the experimental error the same solubility and specific activity. A solution saturated with the crystals of either one of the trypsin preparations does not show any increase in protein concentration or activity when crystals of the other trypsin preparation are added. 8. The Penicillium mold kinase has a slight activating effect on chymo-trypsinogen the rate being only 1-2 per cent of that of trypsinogen. The activation, as in the case of trypsinogen, takes place only in an acid medium. 9. Mold kinase is rapidly destroyed when brought to pH 6.5 or higher, and also when heated to 70 degrees C. In the temperature range of 50-60 degrees C. the inactivation of kinase follows a unimolecular course with a temperature coefficient of Q(10) = 12.1 and micro = 53,500. The molecular weight of mold kinase, as determined by diffusion, is 40,000.
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PMID:FORMATION OF TRYPSIN FROM TRYPSINOGEN BY AN ENZYME PRODUCED BY A MOLD OF THE GENUS PENICILLIUM. 1987 69


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