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:3.1.1.7 (acetylcholinesterase)
28,390 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 (EC 3.1.1.7) has been shown to possess an intrinsic peptidase activity. [Chubb et al. (1983), Neuroscience 10, 1369-1383]. To examine this activity further, the breakdown of a model hexapeptide (leu-trp-met-arg-phe-ala) LWMRFA was studied. Affinity-purified eel acetylcholinesterase rapidly cleaved the hexapeptide in a trypsin-like manner to produce two peptides (LWMR and FA). Acetylcholinesterase more slowly cleaved the C-terminal alanine residue from the peptide to yield LWMRF. Although the enzyme showed preference for cleaving the hexapeptide at its C-terminal, it was also able to cleave the N-terminal leucine residue form the tryptic product LWMR. Hydrolysis of the peptide at the tryptic site (arg4-phe5) was strongly inhibited by the trypsin inhibitor diisopropylfluorophosphate. Cleavage of the C-terminal alanine was only poorly inhibited by diisopropylfluorophosphate, but more strongly inhibited by metal-ion chelating agents, and it was increased in the presence of Zn2+ and Co2+. The pH optimum for cleavage at the tryptic site was 6, while that for the carboxypeptidase site was 8-9. These results show that acetylcholinesterase can hydrolyse peptides like a trypsin-like endopeptidase and a Zn2+- or Co2+-dependent exopeptidase, and they suggest that these two peptidase activities are associated with two separate active sites on the acetylcholinesterase molecule. As both peptidase activities eluted with acetylcholinesterase from a TSK 4000SW column when it was chromatographed by high-performance liquid chromatography, it is unlikely that the presence of either peptidase activity could be attributable to a contaminant in the acetylcholinesterase preparation. We suggest that acetylcholinesterase may be involved in the breakdown of bioactive peptides or their precursors in neuroendocrine cells.
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PMID:Acetylcholinesterase exhibits trypsin-like and metalloexopeptidase-like activity in cleaving a model peptide. 330 51

We have isolated a COOH-terminal tryptic peptide from the hydrophobic globular (5.6 S) form of Torpedo californica acetylcholinesterase that exhibits divergence in amino acid sequence from the catalytic subunit of the dimensionally asymmetric (17 S + 13 S) enzyme. The divergent peptide could be recovered from the glycophospholipid-modified 5.6 S enzyme only after treatment with phosphatidylinositol-specific phospholipase C. Upon reduction, carboxymethylation with [14C]iodoacetate, and trypsin digestion the resultant peptides were purified by gel filtration followed by high performance liquid chromatography. The high performance liquid chromatography profiles of 14C-labeled cysteine peptides from lipase-treated 5.6 S enzyme revealed unique radioactive peaks which had not been present in digests of the asymmetric form. These peaks all yielded identical amino acid sequences. The difference in chromatographic behavior of the individual peptides most likely reflects heterogeneity in post-translational processing. Gas-phase sequencing and composition analysis are consistent with the sequence: Leu-Leu-Asn-Ala-Thr-Ala-Cys. Composition includes 2-3 mol each of glucosamine and ethanolamine which is indicative of modification by glycophospholipid. Glucosamine is also present in an asparagine-linked oligosaccharide. The two forms of acetylcholinesterase diverge after the threonine residue within this peptide sequence; the hydrophobic form terminates with cysteine whereas the asymmetric form extends for 40 residues beyond the divergence. The locus of divergence and absence of any other amino acid sequence difference suggest that the molecular forms of acetylcholinesterase arise from a single gene by alternative mRNA processing.
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PMID:Divergence in primary structure between the molecular forms of acetylcholinesterase. 333 34

A factor that stimulates the incorporation of 75Selenomethionine into the newly formed platelets of recipient mice (thrombopoietin, TPO) has been partially purified from the plasma of thrombocytopenic patients. The activity was precipitated at 60-80% ammonium sulfate saturation and further purified with hydrophobic interaction chromatography. Thrombopoietin was retained by concanavalin-A-Sepharose. Using HPLC size-exclusion chromatography, an approximate molecular weight of 40,000 dalton was calculated. The overall purification factor was about 2,100-fold. TPO was stable in a pH range from 5 to 9 and was heat-sensitive, and the biological activity was destroyed by trypsin treatment and by dithiothreitol. The partially purified molecule did not stimulate the proliferation of megakaryocyte progenitors in vitro and had no effect on the growth of erythroid or granulocyte-macrophage colonies; when administered in-vivo, TPO significantly affected the mean platelet volume and increased the number of small acetylcholinesterase cells in the bone marrow. TPO appears to be specific for the megakaryocytic lineage and active on the postmitotic compartment of megakaryocytes.
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PMID:Partial purification and biochemical characterization of human plasma thrombopoietin. 336 51

In addition to its ability to hydrolyze acetylcholine, purified eel acetylcholinesterase possesses a trypsin-like endopeptidase activity. The tryptic activity is associated with a serine residue at a site that is distinct from the esteratic site. To label both the esteratic and tryptic sites, the enzyme was incubated with the serine hydrolase inhibitor [3H]diisopropyl fluorophosphate. This compound labelled the protein in a biphasic manner, with both slow and rapid labelling kinetics. The time course of the rapid phase was similar to the time course of inactivation of the esteratic activity. The time course of the slow phase was similar to the time course of inactivation of the tryptic activity. Labelling of the nonesteratic site was inhibited by the trypsin inhibitor N alpha-p-tosyl-L-lysine chloromethyl ketone. The total number of sites labelled by [3H]diisopropyl fluorophosphate on eel acetylcholinesterase was 2.6 mol/280,000 g protein, whereas the number of tryptic sites was less (0.52 mol/280,000 g). The results suggest that a subpopulation of acetylcholinesterase molecules may possess tryptic activity. Extensive chromatography of the purified enzyme by ion-exchange and gel filtration failed to separate the labelled tryptic component from acetylcholinesterase. On sodium dodecyl sulfate-polyacrylamide gels, the labelled tryptic component comigrated with a polypeptide of 50,000 molecular weight, which is a major proteolytic digestion product derived from the intact acetylcholinesterase monomer. Because of its localization in many noncholinergic peptide-containing cells, acetylcholinesterase could act as a neuropeptide processing enzyme in these cells.
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PMID:Identification of a trypsin-like site associated with acetylcholinesterase by affinity labelling with [3H]diisopropyl fluorophosphate. 337 13

A previous study of cholinergic development indicated a possible trophic relationship between the olfactory bulb and its afferents from the basal forebrain (Large et al., J. Neurochem., 46 (1986) 671-680). To examine this possibility further, cultured embryonic basal forebrain neurons from rat were used as a test system for trophic factor activity hypothesized to be present in olfactory bulb. Basal forebrain neurons grown in defined medium typically died within 2-3 days. However, survival and differentiation were strikingly enhanced by soluble extracts of olfactory bulb tissue. This trophic effect was noticeable with 2 micrograms/ml olfactory bulb protein, and plateaued at 100 micrograms/ml. The activity was heat- and trypsin-sensitive, non-dialyzable, stable in the cold, resistant to NGF antiserum, and approximately 100-150 kDa in size. Nerve growth factor, bovine serum albumin, laminin and extracts from heart did not mimic the activity. Long-term growth (21 days) in the presence of olfactory bulb proteins resulted in extensive neurite production, formation of thick neurite fascicles, and aggregation of cells. Some glia were present, as evidenced by the presence of glial fibrillary acidic protein, and large numbers of cells were positive for neuron-specific enolase and true acetylcholinesterase. Trophic activity was also present in medium conditioned by olfactory bulb slices, implying secretion of active factors.
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PMID:Soluble proteins from rat olfactory bulb promote the survival and differentiation of cultured basal forebrain neurons. 340 3

The relationship between chemical modifications of arginine derivatives and inhibitory activity to horse serum cholinesterase (BuChE) was investigated. It provided a new insight into the topography of the active site of BuChE. 1) BuChE has the hydrophobic binding pocket, the depth of which corresponds to the length of ethylpiperidine. 2) In the opposite side to the hydrophobic binding pocket, BuChE has a certain entity which repulses carboxyl group at the 2-position of piperidine of L-arginine piperidine amide. 3) The P site of BuChE can allow 4-propyl and 4-phenyl group attached to piperidine. Comparison of the results with those of thrombin and trypsin clearly revealed similarities and dissimilarities among BuChE, trypsin, and thrombin in the active site topography, and hence, we introduce a new selective inhibitor for BuChE, N alpha-dansyl-L-arginine 4-phenylpiperidine amide. It inhibits BuChE strongly (Ki = 0.016 microM), whereas it inhibits trypsin, thrombin, plasmin, and glandular kallikrein only weakly and shows actually no inhibition on acetylcholinesterase from the human erythrocyte. In addition, the new inhibitor becomes highly fluorescent when bound with BuChE, indicating that the compound is an ideal probe of the interactions of BuChE as well as a titrant of it.
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PMID:N alpha-dansyl-L-arginine 4-phenylpiperidine amide. A potent and selective inhibitor of horse serum cholinesterase. 340 26

The currently available radioimmunoassays of trypsin measure total immunoreactive trypsin (EC 3.1.1.7), which includes both trypsinogen and alpha 1-protease inhibitor-bound trypsin. Hitherto, the only way to differentiate these two forms of trypsin has been to fractionate them on a gel-filtration column. We describe here a solid-phase immunoenzymometric assay that rapidly measures the amount of cationic trypsin bound to alpha 1-protease inhibitor in the plasma of rats with experimental pancreatitis. The assay specifically measures this complex within the range from 0.2 to 5.0 ng without interference by high concentrations of free alpha 1-protease inhibitor. The high correlation (r = 0.985) of the values obtained by size fractionation and by this assay demonstrates the accuracy of the assay, which is the first single-tube method for determining this form of activated cationic trypsin in plasma.
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PMID:Immunoenzymometric determination of trypsin/alpha 1-protease inhibitor complex in plasma of rats with experimental pancreatitis. 352 80

The major soluble protein of bovine chromaffin granules chromogranin A was purified by reverse-phase high performance liquid chromatography. Brief incubations with either acetylcholinesterase or trypsin cleaved chromogranin A to yield two chromogranin-immunoreactive polypeptides which were similar in molecular weight to two of the major endogenous chromogranin polypeptides. A number of peptidase inhibitors which strongly inhibited tryptic digestion of chromogranin A also inhibited the acetylcholinesterase digestion, although they were less potent. More prolonged digestion of chromogranin A with acetylcholinesterase produced a large number of peptides which were similar to some of the endogenous chromogranin peptides in their elution profile by high performance liquid chromatography. In contrast, complete tryptic digestion of chromogranin A yielded peptides with a totally different elution profile. The experiments indicate that acetylcholinesterase possesses a peptidase activity which is similar, but not identical to trypsin, and suggest that a second non-tryptic activity is also present. They also suggest that acetylcholinesterase, an enzyme found in chromaffin cells, may process chromogranin A to yield lower molecular weight chromogranins in bovine chromaffin cells.
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PMID:Acetylcholinesterase hydrolyses chromogranin A to yield low molecular weight peptides. 353 42

Proteolytic action on human serum cholinesterase, a tetrameric enzyme, results in a partial disintegration which can be recorded only qualitatively by time-consuming electrophoretic techniques. In this study, a rapid high-performance liquid chromatographic method was used for the separation and determination of the active dissociation products. Separation of the cholinesterase subunits was accomplished by high-performance gel permeation chromatography on a combination of DIOL columns (Zorbax GF 450/GF 250) in 0.2 M phosphate buffer (pH 7.0). Detection and quantification of enzyme activity in the fractionated eluate were carried out using a Flexigem analyser (substrate, butyrylthiocholine). On limited tryptic digestion of partially purified human ChE, up to three peaks of enzyme activity could be identified. Their elution volumes corresponded to apparent molecular masses of 480,000, 270,000 and 120,000, indicating, in addition to the tetrameric holoenzyme, a dimeric and a monomeric form. Quantification of the relative amounts of individual enzyme activity peaks revealed that in the course of degradation, the dimer appeared first, followed by the monomer. This suggests that the first step in the sequence of dissociation is cleavage of the tetramer into a pair of dimers, then further into the monomeric subunit. During the incubation with trypsin, a significant change in the pattern of the different peaks had already occurred when the total enzyme activity was only slightly reduced.
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PMID:Quantitative monitoring by high-performance liquid chromatography of the dissociation of human serum cholinesterase by limited proteolysis. 366 27


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