Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The amyloid protein precursor (APP) of Alzheimer's disease was found to bind saturably (Kd = 60 nM) to embryonic chick brain extracellular matrix (ECM). The binding of APP to ECM was not inhibited by 10 micrograms/ml heparin or heparan sulfate. However, pretreatment of cells with 1 mM 4-methylumbelliferyl-beta-D-xyloside, an inhibitor of proteoglycan biosynthesis, reduced the number of APP binding sites on the ECM by 80%. The binding of APP to ECM was also inhibited by pretreatment with chlorate, an inhibitor of glycan sulfation, and heparitinase, which digests the carbohydrate component of heparan sulfate proteoglycans. These results suggest that APP binds with high affinity to one or more heparan sulfate proteoglycans. Acidic and basic fibroblasts growth factor (FGF) also bound to chick ECM. When ECM was incubated with a protease associated with the enzyme AChE (AChE-AP), APP and acidic FGF were released intact from the matrix. The AChE-AP was at least 100-fold more potent in releasing APP from ECM than other trypsin-like proteases (trypsin, plasmin, thrombin). The action of the AChE-AP was inhibited by glia-derived nexin (protease nexin I) and by human brain APP at low nanomolar concentrations. These results suggest that in vivo an AChE-AP may cleave ECM proteins to regulate the availability of soluble APP or other factors bound to the ECM.
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PMID:Association and release of the amyloid protein precursor of Alzheimer's disease from chick brain extracellular matrix. 127 36

Electricus electrophorus acetylcholinesterase (AChE, EC 3.1.1.7) is reported to possess a trypsin-like activity. We found that purification of AChE removes over 99% of this protease activity, which resides in a single 25 kDa protein with an N-terminal sequence identical to bovine pancreatic trypsin. Digests of neuropeptides using purified eel AChE or bovine pancreatic trypsin gave identical peptide maps. These results indicate that the commercial preparation of eel AChE is contaminated by a trypsin, which is difficult to remove completely during AChE purification.
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PMID:Identification of the trypsin-like activity in commercial preparations of eel acetylcholinesterase. 175 64

Acetylcholinesterase was purified from the soluble supernatant of monkey (Macaca radiata) brain basal ganglia by a three-step affinity purification procedure. The purified enzyme showed two major protein bands corresponding to molecular weights of approximately 65 kDa and approximately 58 kDa which could be labelled by [3H]diisopropylfluorophosphate. When the purified enzyme was subjected to limited trypsin digestion followed by gel filtration on Sephadex G-75 or Sephadex G-25 column, a peptide fragment of molecular weight approximately 300 Da having a weak acetylthiocholine hydrolysing activity was isolated. The amino acid sequence analysis of this peptide showed a sequence of Gly-Pro-Ser. When the [3H]DFP labelled enzyme was subjected to limited trypsin digestion and Sephadex G-75 column chromatography, a labelled peptide corresponding to approximately 430 Da was isolated. The kinetics, inhibition characteristics and binding characteristics to lectins of this peptide were compared with the parent enzyme. A synthetic peptide of sequence Gly-Pro-Ser was also found to exhibit acetylthiocholine hydrolysing activity. The kinetics and inhibition characteristics of the synthetic peptide were similar to those of the peptide derived from the purified acetylcholinesterase, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 123700 times less than that of the purified AChE.
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PMID:Isolation of a tripeptide showing weak acetylthiocholine hydrolysing activity from a soluble form of monkey basal ganglia acetylcholinesterase by limited trypsin digestion. 187 67

The monoclonal antibody (mAb) 2G8 (subclass IgG2a) raised against acetylcholinesterase (AChE, EC 3.1.1.7) from electric organ of Torpedo nacline timilei crossreacted with AChE from Torpedo marmorata, electric eel (Electrophorus electricus), flounder (Platichthys flesus) body muscle, rat brain, bovine brain, and human brain, this suggests that the epitope to which mAb 2G8 bound had been highly conserved during evolution. No crossreaction was found with AChE from human and bovine erythrocytes, nor with butyrylcholinesterase (BtChE, EC 3.1.1.8) from human serum. Binding of mAb 2G8 to the globular G2 form of AChE from T. marmorata strongly decreased enzyme activity, while no significant inhibition was found with either collagen-tailed, asymmetric forms, or with the enzymes from flounder body muscle or mammalian sources. The possibility that mAb 2G8 bound to anionic sites of AChE could be excluded since neither edrophonium chloride nor decamethonium bromide influenced the binding of 2G8 to the enzymes. Enzyme-linked immunosorbent assay and Western blot showed that heat-denatured, diisopropylfluorophosphate-treated, CNBr- and trypsin-digested AChE from T. marmorata still reacted with mAb 2G8; this indicates that the epitope to which 2G8 bound, at least partially, belonged to a continuous determinant. Treatment of cholinesterases with N-glycosidase F abolished crossreaction with 2G8, showing that an essential part of the epitope consisted of N-linked carbohydrates.
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PMID:The monoclonal antibody 2G8 is carbohydrate-specific and distinguishes between different forms of vertebrate cholinesterases. 204 Feb 91

Choline acetyltransferase (Acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6, abbreviated ChAT), the biosynthetic enzyme for acetylcholine and acetylcholinesterase (EC 3.1.1.7, abbreviated AChE) are expressed in a human cholinergic neuroblastoma cell line, MC-IXC. We have shown that ChAT activity can be regulated in culture by retinoic acid, an active metabolite of vitamin A, and by sodium butyrate, an organic fatty acid. Optimal concentrations of these agents produce 4.3-fold and 1.6-fold increases in ChAT activity, respectively. The effects of retinoic acid are statistically significant after 24 h, whereas for sodium butyrate significant differences are seen only after 48 h. Since retinoic acid stimulation of ChAT activity was reversed only by trypsin treatment and not by removal of retinoic acid from the medium, this suggests that this agent may be acting at the level of the cell surface. Other differentiating conditions, such as culture in serum-free medium or addition of 1-2% dimethylsulfoxide did not increase ChAT activity. Acetylcholinesterase activity was shown to increase only in the presence of sodium butyrate, suggesting that retinoic acid and sodium butyrate may be acting via different pathways. Retinoic acid and sodium butyrate both seem to be permissive rather than instructive in regulating ChAT activity in that they are unable to induce ChAT expression de novo in cell lines which do not already express ChAT activity.
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PMID:Stimulation of choline acetyltransferase activity by retinoic acid and sodium butyrate in a cultured human neuroblastoma. 292 23

Acetylcholinesterase (AChE, EC 3.1.1.7) purified from the electric organ of eel possesses a protease activity resembling that of a neuropeptide processing enzyme. To examine whether any mammalian AChEs possess a similar protease activity, the enzyme was purified, 110,000-fold from foetal bovine serum. Purified serum AChE cleaved 2 synthetic peptide substrates in a manner resembling the combined actions of trypsin-like and carboxypeptidase B-like enzymes. A synthetic fragment of preproenkephalin A (residues 97-107) containing a complete methionine-enkephalin sequence was cleaved by serum AChE to yield free methionine-enkephalin. The carboxypeptidase action of AChE was weakly stimulated by the presence of 100 microM CoCl2 suggesting the requirement of a metal ion for complete activity. The results support the hypothesis that in many tissues AChE may act as a neuropeptide processing enzyme.
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PMID:Serum acetylcholinesterase possesses trypsin-like and carboxypeptidase B-like activity. 322 17

Two-site methods were developed for immunoassay of acetylcholinesterase (AChE; EC 3.1.1.7) in crude extracts of rat and human tissues. A radiometric assay for human AChE utilized a specific monoclonal AChE antibody adsorbed to polystyrene microtiter wells at alkaline pH. AChE bound strongly to this antibody after 24 h at 4 degrees C. Bound enzyme was detected with an 125I-labeled antibody against a different AChE epitope. The assay signal was quasi-linearly related to AChE concentration in purified and crude samples, with a detection threshold near 100 pg. Tetrameric and dimeric AChE behaved equivalently in the assay. Two-site methods with a different pair of species-selective antibodies worked equally well for immunoassay of rat AChE. Assays of the rat enzyme showed that immunoreactivity was lost as rapidly as enzyme activity during heating to 54 degrees C. On the other hand, immunoreactivity was preserved despite loss of enzyme activity after exposure to anticholinesterases or trypsin. A biotinylated second antibody detected by alkaline-phosphatase-conjugated avidin was used to develop an AChE enzyme-linked immunosorbent assay (ELISA) with a sensitivity similar to that of the radiometric assay. Either the ELISA or the radiometric immunoassay may be useful whenever proteolysis or other mechanisms are suspected of dissociating enzyme activity and immunoreactivity. In denervated muscle and ligated peripheral nerve, application of the two-site method showed closely parallel variations in immunoreactivity and enzyme activity.
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PMID:Two-site immunoassay for acetylcholinesterase in brain, nerve, and muscle. 329 48

Acetylcholinesterase was purified by passage through 3 affinity columns. The enzyme so purified was found to be homogeneous by electrophoresis and the peptidase and AChE activities co-eluted from a high pressure liquid chromatography column. The purified AChE degraded the chromogranins, the soluble proteins from the adrenal chromaffin granules, at a rate of nearly 8 micrograms/microgram AChE/h. The rate was fastest with the largest chromogranins, but proteins across the whole molecular weight spectrum were hydrolyzed. Immunoassay of extracts after incubation with AChE showed that enkephalin-like material had been produced. Incubations were also done with chromogranins that had been fractionated by size exclusion chromatography. The AChE degraded protein in all fractions and generated enkephalin-like immunoreactive material in fractions where it was produced by sequential treatment with trypsin and carboxypeptidase B. It seems likely, therefore, that AChE can hydrolyze some of the enkephalin precursors that are sensitive to trypsin and carboxypeptidase B, but the one-step nature of its action suggests a mode of action with fewer restrictions. It is concluded that AChE can hydrolyze proteins of widely differing sizes and the data add to the evidence that AChE is able to hydrolyze enkephalin precursors resulting in the generation of immunoreactive peptide.
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PMID:Acetylcholinesterase generates enkephalin-like immunoreactivity when it degrades the soluble proteins (chromogranins) from adrenal chromaffin granules. 352 46

Acetylcholinesterase (EC 3.1.1.7.; AChE) and butyrylcholinesterase (EC 3.1.1.8.; BuChE) from chicken muscle exist as sets of structurally homologous forms with very similar properties. The collagenase sensitivity and aggregation properties of the 'heavy' forms of both enzymes indicate that they possess a collagen-like tail, and their stepwise dissociation by trypsin confirms that they correspond to triple (A12) and double (A8) collagen-tailed tetramers. In addition to this dissociating effect, trypsin digests an important fraction of the catalytic units of AChE, in a progressive manner, removing as much as 30% of the enzyme's mass, without inactivation of the tetramers and of the tailed molecules. The trypsin-modified AChE forms closely resemble the corresponding mammalian AChE forms in their hydrodynamic properties. It is not known whether the trypsin-digestible peptides, which do not appear to be involved in the ionic or hydrophobic interactions of the enzymes, are a fragment of the catalytic subunit or whether they constitute distinct polypeptides.
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PMID:The quaternary structure of chicken acetylcholinesterase and butyrylcholinesterase; effect of collagenase and trypsin. 625 92

We have distinguished three fractions of acetylcholinesterase (AcChoE; acetylcholine acetylhydrolase, EC 3.1.1.7) from Torpedo marmorata electric organs, according to their solubilization characteristics. The low-salt-aggregating collagen-tailed forms are soluble in high-salt buffers; their hydrodynamic properties ae not modified in the presence of detergents. They constitute the A fraction, which amounts to about a third of the tissue's AcChoE activity. The low-salt-soluble (LSS) and detergent-soluble (DS) fractions are not sensitive to ionic strength and collagenase. In the presence of nonionic detergents or bile salts, both fractions behave as a monodisperse "6.3S" form, the properties of which have been investigated mostly in the case of Triton X-100. Disulfide bond reduction dissociates the detergent form into a smaller "5S" form. These two forms are thought to be, respectively, detergent-associated dimers and monomers. In the absence of detergent, the LSS fraction is polydisperse: it contains a major 8S component, 11S and 14S components, and faster-sedimenting aggregates, which appear to represent dimers, tetramers, and higher polymers. The heterogeneity of the 8S component in gel filtration suggests that it also contains variable noncatalytic elements. Upon removal of the detergent the DS fraction forms ill-defined aggregates. Trypsin induces quaternary rearrangements of part of the 8S component into 11S and 14S components, which are still convertible into the detergent form; therefore trypsin probably digests noncatalytic elements. Pronase and proteinase K, on the other hand, convert the enzyme into a dimeric form, G2, that does not interact with detergents, probably by cleaving a minor fragment of the subunit that is involved in hydrophobic interactions.
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PMID:Collagen-tailed and hydrophobic components of acetylcholinesterase in Torpedo marmorata electric organ. 693 97


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