Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Aminopeptidase A (glutamyl aminopeptidase; EC 3.4.11.7) has been cloned from porcine brain and kidney cortex cDNA libraries and the complete primary sequence of the enzyme deduced. This predicts a type II integral membrane protein of 942 amino acids with 14 potential N-linked glycosylation sites and a His-Glu-Xaa-Xaa-His zinc binding motif. Aminopeptidase A was purified from porcine kidney cortex by a combination of anion exchange and hydrophobic interaction chromatographies following its release from the membrane by trypsin. The purified protein migrated as three major polypeptides on SDS-polyacrylamide gel electrophoresis of M(r) 147,000, 107,000, and 45,000. N-Terminal sequencing revealed that both the Mr 147,000 and 107,000 polypeptides had the same N-terminal sequence resulting from cleavage of aminopeptidase A by trypsin at the Lys-42-Asp-43 bond just outside the membrane-spanning hydrophobic region. Immunoelectrophoretic blot analysis following electrophoresis under nonreducing conditions revealed that the trypsin-cleaved form of the enzyme no longer migrated as a disulfide-linked dimer, placing the interchain disulfide link N-terminal to Lys-42. N-Terminal sequencing of the M(r) 45,000 polypeptide in the purified preparation of aminopeptidase A revealed that it resulted from cleavage at the Asn-602-Gly-603 bond by an endogenous protease. This posttranslational proteolytic cleavage occurred in porcine kidney cortex microvillar membranes but not in porcine intestinal microvillar membranes. Incubation of purified porcine kidney aminopeptidase N (membrane alanyl aminopeptidase; EC 3.4.11.2) with trypsin resulted in a similar fragmentation pattern to that observed in aminopeptidase A, suggesting that these and other members of the type II membrane-spanning zinc aminopeptidase family may have two distinct domains: an N-terminal domain, containing the zinc binding site and residues identified as being involved in catalysis, and a C-terminal domain of unknown function, that are separated by a protease-susceptible region.
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PMID:Proteolytic fragmentation reveals the oligomeric and domain structure of porcine aminopeptidase A. 906 31

We have been developing a novel bioadhesive drug-carrier matrix that protects embedded therapeutic peptides and proteins from degradation by the most abundant intestinal proteases. Increasing amounts of the Bowman-Birk inhibitor (BBI) were thereby covalently linked to chitosan-EDTA. The bioadhesive properties of the resulting polymer-BBI conjugates and their inhibitory effect toward trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), elastase (3.4.21.36), carboxypeptidase A (EC 3.4.17.1), and aminopeptidase N (EC 3.4.11.2) were evaluated in vitro. Whereas unmodified chitosan-EDTA exhibited under our experimental conditions an adhesive strength of 54.4 +/- 7.7 mN, it was determined to be 21.0 +/- 3.8 mN for the comparably most adhesive polymer-BBI conjugate (mean +/- SD; n = 5). All polymer-BBI conjugates showed a strong inhibitory activity toward the serine proteases trypsin and chymotrypsin. However, the protective effect toward elastase was markedly lower. Due to the high binding affinity of chitosan-EDTA toward zinc, which represents an essential cofactor for carboxypeptidase A and aminopeptidase N, all polymer-BBI conjugates displayed additionally a strong protective effect toward these exopeptidases. The novel bioadhesive polymer-BBI conjugates described in this study seem to be very useful drug-carrier matrixes in overcoming the enzymatic barrier to orally administered peptide and protein drugs.
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PMID:Intestinal peptide and protein delivery: novel bioadhesive drug-carrier matrix shielding from enzymatic attack. 954 94

The purpose of the present study was to synthesize and evaluate mucoadhesive polymers, exhibiting a high capacity to bind bivalent cations which are essential co-factors for intestinal proteolytic enzymes. Under the formation of amide bonds, the complexing agent EDTA was covalently bound to the primary amino groups of chitosan. One gram of the resulting conjugate with the lowest amount of remaining free amino groups (0.1 +/- 0.03%; mean +/- SD, n = 3) based on free chitosan as 1.0 was capable of binding 1.4 +/- 0.1 mM calcium, 2.0 +/- 0.1 mM zinc and 1.9 +/- 0.03 mM cobalt (mean +/- SD, n = 3) under intestinal pH-conditions, respectively. Whereas proteolytic activity of the serine proteases trypsin (EC 3.4.21.4), alpha-chymotrypsin (EC 3.4.21.1) and elastase (EC 3.4.21.36) could not be inhibited, proteolytic activity of the zinc proteases carboxypeptidase A (EC 3.4.17.1) and aminopeptidase N (EC 3.4.11.2) was strongly inhibited by the chitosan-EDTA conjugate. Moreover, it displays quick swelling properties in water and basic aqueous solutions. The adhesive force of the conjugate was even higher than of chitosan HCl. However, lowering the percentage of covalently attached EDTA on the polymer, leads to a significantly reduced adhesive force. According to these results, chitosan-EDTA conjugates exhibiting the lowest amount of remaining free amino groups, seem to be a useful tool in overcoming the enzymatic barrier for perorally administered therapeutic peptides.
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PMID:Mucoadhesive polymers as platforms for peroral peptide delivery and absorption: synthesis and evaluation of different chitosan-EDTA conjugates. 968 88

The metabolism of three opioid tetrapeptides, Tyr-D-Arg-Phe-Nva-NH2, Tyr-D-Arg-Phe-Phe-NH2 and Tyr-D-Ala-Phe-Phe-NH2, was investigated in the presence of pure pancreatic enzymes (trypsin, chymotrypsin, elastase, carboxypeptidase A and carboxypeptidase B), as well as in the presence of pure carboxylesterase and aminopeptidase N. The cleavage patterns of the pure pancreatic enzymes were then compared with those found in rat and human jejunal fluid. Metabolism was also studied in homogenates from different intestinal regions (duodenum, jejunum, ileum and colon) and in enterocyte cytosol from rats. The effect of various protease inhibitors was investigated in the jejunal homogenate. The parent peptides were assayed by high-performance liquid chromatography and metabolites were identified by means of liquid chromatography-mass spectrometry. Of the pure enzymes, the quickest hydrolysis of the peptides was observed for the pancreatic enzymes chymotrypsin, trypsin and carboxypeptidase A. In most cases they formed the corresponding deamidated tetrapeptides (chymotrypsin and trypsin) or tripeptides with a missing C-terminal amino acid (carboxypeptidase A). Regional differences in intestinal metabolism rates were found for all three peptides (P < 0.001), with the highest rates observed in jejunal and/or colonic homogenates. The deamidated tetrapeptides were formed both in rat intestinal homogenates and in enterocyte cytosol. Metabolism in the jejunal homogenate was markedly inhibited by some serine and combined serine and cysteine protease inhibitors. In conclusion, the C-terminal amide of these tetrapeptides did not fully stabilise them against intestinal deamidase and carboxypeptidase activities. The significant hydrolysis of the peptides by pure chymotrypsin, trypsin and carboxypeptidase A showed that lumenal pancreatic proteases might be a clear metabolic obstacle in oral delivery even for small peptides such as these tetrapeptides.
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PMID:Investigations of the in-vitro metabolism of three opioid tetrapeptides by pancreatic and intestinal enzymes. 1093 29

In rheumatic joints, high concentrations of interleukin-8 (IL-8) have been measured in synovial fluid and in pannus tissue. In both locations aminopeptidase N (APN)/CD13, an exopeptidase with reported activity towards IL-8 is also present. The surprising stability of IL-8 in the presence of an alleged IL-8-degrading peptidase prompted us to undertake the present study. Cocultivation of fibroblast-like synoviocytes (SFC) with T cells or with T lymphocytic cell membranes, or of T cells with SFC cell membranes, all resulted in increased IL-8 mRNA expression and IL-8 secretion into the medium, and an increase of APN expression on lymphocytes. IL-8 degradation was monitored by Western blots and HPLC. IL-8(72), as a partially processed form, was used throughout this study since it is abundant in tissues and has increased biological activity in comparison to IL-8(77). Thus its degradation/inactivation is considered of high biological significance. Whereas trypsin as a positive control rapidly degraded IL-8, we did not see any IL-8 degradation, either by a variety of soluble APNs, by leucine aminopeptidase or by APN expressed on the surface of SFC, or on ECV304 cells transfected with an APN expression vector. The much more sensitive HPLC technique resulted in negative results as well.
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PMID:Increased expression of interleukin-8 and aminopeptidase N by cell-cell contact: interleukin-8 is resistant to degradation by aminopeptidase N/CD13. 1139 21

Cry1Ac from Bacillus thuringiensis ssp. kurstaki HD-73 is a pore-forming protein specifically toxic to lepidopteran insect larvae. It binds to the cell-surface receptor aminopeptidase N in Manduca sexta midgut via the sugar N-acetyl-D-galactosamine (GalNAc). By using 1,3-diaminopropane (DAP) as the buffer throughout protoxin activation and chromatography on Q-Sepharose at pH 10.3, trypsin-activated Cry1Ac has been purified in a monomeric state, which was crucial to obtaining single crystals of Cry1Ac and of the Cry1Ac-GalNAc complex. Crystals of Cry1Ac alone are triclinic, with unit-cell parameters a = 51.78, b = 113.23, c = 123.41 A, alpha = 113.11, beta = 91.49, gamma = 100.46 degrees; those of the Cry1Ac-GalNAc complex show P2(1) symmetry, with unit-cell parameters a = 121.36, b = 51.19, c = 210.56 A, beta = 105.75 degrees. Data sets collected to 2.36 and 2.95 A resolution, respectively, show that both crystal forms contain four molecules of the 66 kDa toxin in the asymmetric unit and have related packing arrangements. The deaggregating effect of DAP may be explained by its capacity for bivalent hydrogen bonding and hydrophobic interactions at protein interfaces.
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PMID:Crystallization of the Bacillus thuringiensis toxin Cry1Ac and its complex with the receptor ligand N-acetyl-D-galactosamine. 1171 24

Marine fish larvae undergo major morphological and cellular changes during the first month of life. The ontogeny of the gastrointestinal tract combines these two aspects of the larval development and is very interesting in that the timing of functional changes appears genetically hard-wired. The goal of this paper is to give an overview of the gastrointestinal development process in marine fish larvae, with particular attention to three species: sea bass; red drum; and sole, since the description of gut maturation in fish larvae was initiated during the last decade with these species. During the early stages, marine fish larvae exhibit particular digestive features. Concerning the exocrine pancreas, amylase expression decreases with age from the third week post-hatching in sea bass and red drum (approximately 400 degree days), whereas expression of other enzymes (trypsin, lipase, phospholipase A2...) increases until the end of the larva period. Moreover, secretory function of the exocrine pancreas progressively develops and becomes efficient after the third week of life. Concerning the intestine, enzymes of the enterocyte cytosol (in particular peptidase) have higher activity in young larvae than in older. Approximately in the fourth week of post-hatching development in sea bass, red drum and sole larvae, the cytosolic activities dramatically decline concurrently with a sharp increase in membranous enzyme activities of the brush border, such as alkaline phosphatase, aminopeptidase N, maltase. This process characterises the normal maturation of enterocytes in developing fish larvae and also in other vertebrates' species. The establishment of an efficient brush border membrane digestion represents the adult mode of digestion of enterocytes. This paper also describes the role of diet on the development of the gastrointestinal tract. Indeed, the maturational process of digestive enzyme can be enhanced, stopped, or delayed depending on the composition of the diet.
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PMID:Ontogeny of the gastrointestinal tract of marine fish larvae. 1173 35

Dynamic light scattering and surface plasmon resonance techniques were used to investigate the influence of ionic strength, buffer composition and pH on the multimerization of trypsin-activated Cry1Ac and Cry1C toxins over time and the subsequent effects of the different multimers on receptor binding models. In carbonate buffer at pH 10.5, Cry1Ac and Cry1C assumed a monomeric state. After 24 h, a complete conversion of monomeric toxin to a dimeric or trimeric form was observed only for Cry1Ac under low ionic strength condition. Cry1C and Cry1Ac in high ionic strength buffer remained monomeric. Substitution of CAPS pH 11 for carbonate buffer suppressed this Cry1Ac oligomerization effect. Once Cry1Ac toxin was in an aggregated form, increases in ionic strength failed to revert the aggregated toxin back to a monomeric form. Monomeric Cry1Ac bound to a purified 115 kDa aminopeptidase N receptor from Manduca sexta in a 2:1 molar ratio thus confirming the existence of two binding sites on this receptor. Binding rates of dimeric or higher aggregated Cry1Ac toxin forms were different from those generated using the monomeric form and could not be fitted to existing binding models. In summary, our results confirm that the M. sexta 115 kDa aminopeptidase N receptor possesses two Cry1Ac binding sites. They further suggest that although high pH and low salt conditions promote Cry1Ac aggregation, this observation cannot be applied universally to other members of the Cry family.
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PMID:Polydispersity of Bacillus thuringiensis Cry1 toxins in solution and its effect on receptor binding kinetics. 1190 22

Maturation of the fetal gastrointestinal tract (GIT) is influenced by both luminal stimuli (e.g. swallowed fluid) and hormonal factors (e.g. endogenous cortisol release). The aims of the present study were 1) to investigate GIT growth and maturation during the last 20% of gestation in pigs (term = 114 +/- 2 d), and 2) to investigate the effect of esophageal ligation, to prevent fetal swallowing, at 80% to 91% gestation. In normal fetuses, marked increases occurred during late gestation in body weight (+95%), relative intestinal weight (+79%, g kg(-1) body weight), activity of some digestive enzymes (1.5- to 10-fold), and absorption of glucose and intact proteins (3- to 6-fold). Fetuses with ligated esophagi had lowered body weight (-20%), reduced intestinal weight (-43%), aminopeptidase A activity (-24%), and glucose absorption (-27%), while lactase, sucrase, and dipeptidylpeptidase IV activities were increased (+40-50%), compared with sham-operated fetuses (all p < 0.05). Other parameters of GIT function remained unchanged by esophageal obstruction (absorption of amino acids and immunoglobulin, activity of chymosin, amylase, trypsin, chymotrypsin, maltase, aminopeptidase N -- all expressed per gram GIT tissue). Ligated fetuses had elevated cortisol levels, which is known to stimulate fetal GIT maturation. We conclude that the rapid development of GIT function in late gestation is diminished by esophageal obstruction, mainly due to slower GIT growth and not inhibition of normal functional development of enterocytes.
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PMID:Prenatal development of gastrointestinal function in the pig and the effects of fetal esophageal obstruction. 1219 78

To simulate the effects of nutritionally adequate and inadequate vegetarian diets, rats were fed, for 28 days, an isonitrogenous, isocaloric, amino acid unbalanced cereal diet (CD) deficient in lysine and tryptophan or a balanced cereal-legume diet (CLD). The impact of these diets on enzymes responsible for digestion of proteins and carbohydrates were measured. Neither experimental diet significantly affected the animal's final weight or feed consumption in comparison with controls fed a standard mixed diet from plant and animal sources. However, during the first three weeks, the weight gain of rats fed the CD was significantly lower (p < 0.01; p < 0.05) than that of the controls. CD fed rats also had a higher feed efficiency ratio (p < 0.05), demonstrating increased feed consumption per unit of body weight. They also had decreased pancreatic alpha-amylase activity (p < 0.05), serum phytolytic and zoolytic alpha-amylase activity (p < 0.05) and serum protein level (p < 0.05) than the controls. Activity of pancreatic trypsin and intestinal enzymes (sucrase, maltase, aminopeptidase N) were the same as in the controls. In rats fed CLD, growth, food consumption, and enzyme activities did not change, however serum protein and glucose levels were higher (p < 0.025; p < 0.005) than in the controls. It is hypothesized that decrease in alpha-amylase activity was mostly related to the tryptophan deficiency in the CD because this enzyme contains the highest amount of tryptophan units among all tested enzymes.
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PMID:Pancreatic and intestinal enzyme activities in rats in response to balanced and unbalanced plant diets. 1260 33


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