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)

Attempts were made to solubilize acetylcholinesterase (AChE) from microsomal membranes isolated from rabbit white muscle. The preparative procedure included a step in which the microsomes were incubated in a solution containing high salt concentration (0.6 M KCl). About 15% of the total enzyme activity could be solubilized with dilute buffer. Addition of EDTA (1 mM), EGTA (1 mM) or NaCl (0.5 and 1 M) to the extraction buffer did not improve the solubilization yield. Several non-ionic detergents and biliary salts were then used to bring the enzyme into solution. Triton X-100, C12E9 (dodecylnonaethylenglycol monoether) and biliary salt, above their critical micellar concentration, proved to be very effective as solubilizing agents. The occurrence of multiple molecular forms in detergent-soluble AChE was investigated by means of molecular sieving, centrifugation analysis, and slab gel electrophoresis. Experiments on gel filtration showed that, during the process, half of the enzyme was transformed into aggregates, the rest of the activity appearing as peaks with Stokes radii ranging from 3.7 to 7.9 nm. Both ionic strength and detergent nature modify the number and relative proportion of these peaks. Centrifugation analysis of Triton-saline-soluble AChE yielded molecular forms of 4.8S, 10-11S, and 13.5S, whereas deoxycholate extracts revealed species of 4.8S, 10S, and 15S, providing that gradients were prepared with 0.5 M NaCl. In the absence of salt, forms of 6.5-7.5S, 10S, and 15S were measured. The lightest species was always the predominant form. Slab gel electrophoresis showed several bands (68,000-445,000). The 4.8S component only yielded bands of 65,000-70,000.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Solubilization and partial characterization of acetylcholinesterase from the sarcotubular system of skeletal muscle. 361 10

The content and distribution of acetylcholinesterase (AChE) activity in the skeletal muscle disorder malignant hyperthermia (MH) was examined. The AChE activity of sarcolemma membranes isolated from MH-susceptible (MHS) swine was increased twofold when compared with normal sarcolemma. The total AChE activity of muscle extracts was also doubled in MHS tissue; however, the relative distribution between low-salt extractable (globular forms) and high-salt extractable (asymmetric forms) AChE activities were similar in MHS and normal muscle. Our results suggest that, for as yet unexplained reasons, both the sarcolemmal and total AChE activity of skeletal muscle is increased in porcine MH.
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PMID:Increased skeletal muscle acetylcholinesterase activity in porcine malignant hyperthermia. 368 45

It is shown that the salt effect in acetylcholinesterase-catalyzed hydrolysis of 2-(N-methylmorpholinium)-ethylacetate can be quantitatively described by the equation log(k2/KS) = log(k2/KS) degrees--psi log[M+Z] following from Manning's polyelectrolyte theory; the psi values for salts with univalent and bivalent cations at different pH values of the reaction medium were in accordance with the conclusions of the theory. Manning's polyelectrolyte theory seems to be a useful framework for studying salt effects in the reactions of charged substrates with enzymes as globular polyions.
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PMID:Acetylcholinesterase as polyelectrolyte in reaction with cationic substrates. 369 7

The resting efflux of choline from perfused chicken hearts varied from 0.4 to 2.6 nmol/g min, but was constant for at least 80 min in the individual experiments. The rate of choline efflux was found to be equal to the rate of choline formation in the heart, which, from the following reasons, was essentially due to hydrolysis of choline phospholipids. Cardiac content of choline phospholipids (7,200 nmol/g) was much higher than that of acetylcholine (5.5 nmol/g). Resting release of acetylcholine was 0.016 nmol/g min and, after inhibition of cholinesterase, only about 0.1 nmol/g min. Resting efflux of choline was reduced by mepacrine, a phospholipase A2 inhibitor, by perfusion with a Ca2+-free Tyrode's solution containing EGTA and by the combination mepacrine plus Ca2+-free/EGTA solution. In all experiments the reduced choline efflux levelled off within 10 min at about 50%. Omission or elevation of Mg2+ from 1.05 to 10.5 mmol/l had no effect. Resting efflux was increased to 150% by oleic acid (as sodium salt; 2 X 10(-5) mol/l) which is known to activate phospholipase D. Likewise muscarinic agonists (carbachol and acetylcholine) caused facilitation of the efflux of endogenous choline that was blocked by 3 X 10(-7) mol/l atropine. This effect was not reduced, but even slightly enhanced, by mepacrine and by infusion of EGTA in a modified Tyrode's solution (Ca2+-free, 10.5 mmol/l Mg2+). It is concluded that the resting efflux of choline from the heart is essentially due to hydrolysis of choline phospholipids, that half of the efflux is insensitive to mepacrine and is Ca2+-independent (excluding an involvement of phospholipase A2).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of choline efflux from the perfused heart at rest and after muscarine receptor activation. 371 69

The mechanism of dealkylation ("aging") of branched-alkyl organophosphonyl conjugates of acetylcholinesterase and the consequence of this reaction on enzyme conformation were examined by employing kinetic, equilibrium, and spectroscopic techniques. Aging of cycloheptyl methylphosphono-acetylcholinesterase proceeded as a unimolecular reaction in which the enzyme became refractory to oxime reactivation and was accelerated with increases in temperature and decreases in pH and ionic strength of the medium. While aging occurred in a manner invariant with the nature of the salt in buffers containing Na+, K+, Rb+, Cs+, Cl-, CH3COO-, SO2-(4), and PO3-(4), the influence of ionic strength on aging was opposite to that predicted for a mechanism requiring charge separation during formation of the polar transition state. Examination of the equilibrium enzyme conformation with decidium, a fluorescent active center-selective ligand, revealed marked alterations in ligand association and a greater ionic strength dependence for binding after aging. The explanation for this behavior focuses on the high net negative surface charge of the enzyme and proposes that acetylcholinesterase topography is governed by the strength of electrostatic interactions between charged, contiguous, mobile protein regions within the subunit. As such, these studies reveal a reciprocal relationship between acetylcholinesterase topography, surface charge, and ionic strength of the medium.
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PMID:Kinetic, equilibrium, and spectroscopic studies on dealkylation ("aging") of alkyl organophosphonyl acetylcholinesterase. Electrostatic control of enzyme topography. 373 23

We have determined partial N-terminal sequences of acetylcholinesterase (AChE) catalytic subunits from Torpedo marmorata electric organs and from bovine caudate nucleus. We obtain identical sequences (23 amino acids) for the soluble ('low-salt-soluble' or LSS fraction) and particulate ('detergent-soluble', or DS fraction) amphiphilic dimers (G2 form) and for the asymmetric, collagen-tailed forms ('high-salt-soluble', or HSS fraction, A12 + A8 forms). There are two amino acid differences, at position 3 (Asp/His) and 20 (Ile/Val), with the sequences obtained for T. californica by MacPhee-Quigley et al. [(1985) J. Biol. Chem. 260, 12185-12189] for the soluble G2 form and the lytic G4 form which is derived from asymmetric AChE. The bovine sequence (12 amino acids) presents an identity of 4 amino acids (Glu-Leu-Leu-Val) with that of Torpedo, at positions 5-8 (Torpedo) and 7-10 (bovine). There is also a clear homology with the sequence of human butyrylcholinesterase [(1986) Lockridge et al. J. Biol. Chem., in press] indicating that these enzymes probably derive from a common ancestor.
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PMID:Identical N-terminal peptide sequences of asymmetric forms and of low-salt-soluble and detergent-soluble amphiphilic dimers of Torpedo acetylcholinesterase. Comparison with bovine acetylcholinesterase. 379 44

Eleven unique monoclonal IgG antibodies were raised against rabbit brain acetylcholinesterase (AChE, EC 3.1.1.7), purified to electrophoretic homogeneity by a two-step procedure involving immunoaffinity chromatography. The apparent dissociation constants of these antibodies for rabbit AChE ranged from about 10 nM to more than 100 nM (assuming one binding site per catalytic subunit). Species cross-reactivity was investigated with crude brain extracts from rabbit, rat, mouse cat, guinea pig, and human. One antibody bound rabbit AChE exclusively; most bound AChE from three or four species; two bound enzyme from all species tested. Identical, moderate affinity for rat and mouse brain AChE was displayed by two antibodies; two others were able to distinguish between these similar antigens. Nine of the antibodies had lowered affinity for AChE in the presence of 1 M NaCl, but two were salt resistant. Analysis of mutual interferences in AChE binding suggested that certain of the antibodies were competing for nearby epitopes on the AChE surface. One antibody was a potent AChE inhibitor (IC50 = 10(-8) M), blocking up to 90% of the enzyme activity. Most of the antibodies were less able to bind the readily soluble AChE of detergent-free brain extracts than the AChE which required detergent for solubilization. The extreme case, an antibody that was unable to recognize nearly half of the "soluble" AChE, was suspected of lacking affinity for the hydrophilic enzyme form.
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PMID:Monoclonal antibodies to rabbit brain acetylcholinesterase: selective enzyme inhibition, differential affinity for enzyme forms, and cross-reactivity with other mammalian cholinesterases. 388 23

The secretion and cellular localization of the molecular forms of acetylcholinesterase (AChE) were studied in primary cultures of rat sympathetic neurons. When cultured under conditions favoring a noradrenergic phenotype, these neurons synthesized and secreted large quantities of the tetrameric G4, and the dodecameric A12 forms, and minor amounts of the G1 and G2 forms. When these neurons adopted the cholinergic phenotype, i.e., in the presence of muscle-conditioned medium, the development of the cellular A12 form was completely inhibited. These neurons secreted only globular, mainly G4, AChE. Both cellular and secreted A12 AChE in adrenergic cultures aggregated at an ionic strength similar to that of the culture medium, raising the hypothesis that this form was associated with a polyanionic component of basal lamina. In noradrenergic neurons, 60-80% of the catalytic sites were exposed at the cell surface. In particular, 80% of G4 form, but only 60% of the A12 form, was external, demonstrating for the A12 form a sizeable intracellular pool. The hydrophobic character of the molecular forms was studied in relation to their cellular localization. As in muscle cells, most of the G4 form was membrane-bound. Whereas 76% of the cell surface A12 form was solubilized in the aqueous phase by high salt concentrations, only 50% of the intracellular A12 form was solubilized under these conditions. The rest of intracellular A12 could be solubilized by detergents and was thus either membrane-bound or entrapped in vesicles originating from, e.g., the Golgi apparatus.
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PMID:Cellular localization of the molecular forms of acetylcholinesterase in primary cultures of rat sympathetic neurons and analysis of the secreted enzyme. 394 12

Heparin solubilizes asymmetric acetylcholinesterase, from chick skeletal muscle and retina, as a 24 S complex which is quantitatively converted to conventional asymmetric molecular forms of the enzyme (A12 and A8, either class I or class II) upon exposure to high salt. The simultaneous presence of salt and heparin in the homogenization medium selectively prevents, however, the release of class II A-forms in both muscle and retina. Heparin may generally act by displacing native proteoglycans involved in the attachment of the enzyme tail to the extracellular matrix, or its neural equivalent, being in turn removed by salt to yield typical asymmetric enzyme forms. Heparin would also appear to displace some other molecules specifically involved in the EDTA-sensitive attachment of class II tailed forms, this effect being antagonized by salt.
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PMID:Heparin and the solubilization of asymmetric acetylcholinesterase. 394 8

We have stabilized and studied choline acetyltransferase from the nematode Caenorhabditis elegans. The enzyme is soluble, and two discrete forms were resolved by gel filtration. The larger of these two forms (MW approximately 154,000) was somewhat unstable and in the presence of 0.5 M NaI was converted to a form indistinguishable from the "native" small form (MW approximately 71,000). We have purified the small form of the enzyme greater than 3,300-fold by a combination of gel filtration, ion-exchange chromatography, and nucleotide affinity chromatography. The purified preparation has a measured specific activity of 3.74 mumol/min/mg protein, and is free of acetylcholinesterase and acetyl-CoA hydrolase activities. The Vmax of the purified enzyme is stimulated by NaCl, with half-maximal stimulation at 80 mM NaCl. The Km for each substrate is also affected by salt, but in different manners from each other and the Vmax; the kinetic parameter Vmax/Km thus changes significantly as a function of the salt concentration.
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PMID:Properties and partial purification of choline acetyltransferase from the nematode Caenorhabditis elegans. 396 27


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