[13] Acetylcholinesterase

Rosenberry, Terrone L.; Barnett, Philip; Mays, Carol

doi:10.1016/0076-6879(82)82070-3

Cited by 34 publications

(8 citation statements)

References 24 publications

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“…RBC AChE was extracted from outdated erythrocytes with Triton X-100, purified by affinity chromatography on acridinium resin, and depleted of Triton X-100 by chromatography on hydroxyapatite as described by Rosenberry & Scoggin (1984). G4 AChE from the eel Electrophorus electricus was prepared by digesting purified 18s plus 14s eel AChE (0.5 mg/mL in 1.0 M NaCl, 5 mM decamethonium bromide, and 20 mM sodium phosphate, pH 7; Rosenberry et al, 1982) with trypsin (1 mg/mL) for 1 h at 25 "C and separating the AChE activity peak corresponding to a Stokes radius (R,) of 8.8 nm on Sepharose CL-4B in 20 mM sodium phosphate, pH 7. Bovine catalase and Escherichia coli 8-galactosidase (R, = 5.2 and 8.2 nm, respectively; Bon et al, 1976) and aqueous suspensions of papain (25-28 mg/mL) were the best grade available from Sigma Chemical * G, refers to a globular AChE form with n catalytic subunits.…”

Section: Methodsmentioning

confidence: 99%

A small hydrophobic domain that localizes human erythrocyte acetylcholinesterase in liposomal membranes is cleaved by papain digestion

Kim¹,

Rosenberry²

1985

Biochemistry

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A small hydrophobic domain in isolated human erythrocyte acetylcholinesterase is responsible for the interaction of this enzyme with detergent micelles and the aggregation of the enzyme on removal of detergent. Papain has been shown to cleave this hydrophobic domain and to generate a fully active hydrophilic enzyme that shows no tendency to interact with detergents or to aggregate [Dutta-Choudhury, T.A., & Rosenberry, T.L. (1984) J. Biol. Chem. 259, 5653-5660]. We report here that the intact enzyme could be reconstituted into phospholipid liposomes while the papain-disaggregated enzyme showed no capacity for reconstitution. More than 80% of the enzyme reconstituted into small liposomes could be released by papain digestion as the hydrophilic form. Papain was less effective in releasing the enzyme from large liposomes that were probably multilamellar. In a novel application of affinity chromatography on acridinium resin, enzyme reconstituted into small liposomes in the presence of excess phospholipid was purified to a level of 1 enzyme molecule per 4000 phospholipid molecules, a ratio expected if each enzyme molecule was associated with a small, unilamellar liposome. Subunits in the hydrophilic enzyme form released from reconstituted liposomes by papain digestion showed a mass decrease of about 2 kilodaltons relative to the intact subunits according to acrylamide gel electrophoresis in sodium dodecyl sulfate, a difference similar to that observed previously following papain digestion of the soluble enzyme aggregates. The data were consistent with the hypothesis that the same hydrophobic domain in the enzyme is responsible for the interaction of the enzyme with detergent micelles, the aggregation of the enzyme in the absence of detergent, and the incorporation of the enzyme into reconstituted phospholipid membranes.

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Section: Methodsmentioning

confidence: 99%

A small hydrophobic domain that localizes human erythrocyte acetylcholinesterase in liposomal membranes is cleaved by papain digestion

Kim¹,

Rosenberry²

1985

Biochemistry

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“…Recently it has been shown that the basic subunit of conglutinin is a single polypeptide chain that is organized into three domains: two non-collagenous domains separated by a collagenlike region (Davis & Lachmann, 1984). Other than the collagens themselves, only conglutinin, Clq (Brodsky-Doyle et al, 1976) and acetylcholinesterase (Rosenberry et al, 1976) are known to contain interspersed collagenous and non-collagenous regions.…”

Section: Introductionmentioning

confidence: 99%

Ultrastructure and composition of bovine conglutinin

Strang¹,

Slayter²,

Lachmann³

et al. 1986

Biochemical Journal

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Conglutinin binds in a Ca2+-dependent manner to the carbohydrate portion of zymosan and cell-bound iC3b (complement subcomponent C3b cleaved by Factor I in the presence of factor H) similarly to lectin-like proteins that participate in the clearance of plasma glycoproteins. This carbohydrate-binding protein has been found to include both collagenous and non-collagenous domains. Electron micrographs of bovine conglutinin are presented in which conglutinin appears as a tetramer of four 'lollipop' structures emanating from a central hub. The stem region, linking each head to the central hub, is quite stiff, whereas the hub-stem junction is a flexible hinge. From electron micrographs of a pepsin digest of conglutinin, the linkage region is identified as the collagenous portion of the macromolecule. Conglutinin is a multimer of a single polypeptide chain. From sedimentation equilibria of unreduced as compared with reduced and alkylated conglutinin, there are determined to be three disulphide-linked chains. These data, combined with information on the subunit polypeptide of conglutinin, suggest a model for conglutinin in which four disulphide-linked trimers are associated via the N-termini to form the intact macromolecule as viewed in the electron microscope. The ultrastructure of conglutinin appears ideally suited to its lectin-like function.

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“…The resulting compound IV was purified by reverse-phase HPLC on a Vydac C-18 column (2.2 ϫ 25 cm). 1 (22) at room temperature with ␤-MTS-propionic acid (2.0 eq) in the presence of diisopropylethylamine (4 eq) and BOP (1.3 eq) in dimethylformamide. The resulting compound V, after work-up, was purified by HPLC as described for compound IV.…”

Section: -[N ␥ -(␤-Mts-propionyl)-␥-aminopropylamino]acridine (Iv)mentioning

confidence: 99%

“…Trifluoroacetate-Compound IV was prepared in 81% yield by reacting 9-␥-aminopropylaminoacridine dihydrobromide (22) at room temperature with ␤-MTS-propionic acid (2-carboxyethyl methanethiosulfonate) (Toronto Research Chemicals, Inc.) (1.2 eq) and diisopropylethylamine (4 eq) in the presence of activating agent BOP (1.3 eq) in N,N-dimethylformamide. The resulting compound IV was purified by reverse-phase HPLC on a Vydac C-18 column (2.2 ϫ 25 cm).…”

Section: -[N ␥ -(␤-Mts-propionyl)-␥-aminopropylamino]acridine (Iv)mentioning

confidence: 99%

Inhibitors Tethered Near the Acetylcholinesterase Active Site Serve as Molecular Rulers of the Peripheral and Acylation Sites

Johnson

Cusack

Hughes

et al. 2003

Journal of Biological Chemistry

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The acetylcholinesterase (AChE) active site consists of a narrow gorge with two separate ligand binding sites: an acylation site (or A-site) at the bottom of the gorge where substrate hydrolysis occurs and a peripheral site (or P-site) at the gorge mouth. AChE is inactivated by organophosphates as they pass through the P-site and phosphorylate the catalytic serine in the A-site. One strategy to protect against organophosphate inactivation is to design cyclic ligands that will bind specifically to the P-site and block the passage of organophosphates but not acetylcholine. To accelerate the process of identifying cyclic compounds with high affinity for the AChE P-site, we introduced a cysteine residue near the rim of the P-site by site-specific mutagenesis to generate recombinant human H287C AChE. Compounds were synthesized with a highly reactive methanethiosulfonyl substituent and linked to this cysteine through a disulfide bond. The advantages of this tethering were demonstrated with H287C AChE modified with six compounds, consisting of cationic trialkylammonium, acridinium, and tacrine ligands with tethers of varying length. Modification by ligands with short tethers had little effect on catalytic properties, but longer tethering resulted in shifts in substrate hydrolysis profiles and reduced affinity for acridinium affinity resin. Molecular modeling calculations indicated that cationic ligands with tethers of intermediate length bound to the P-site, whereas those with long tethers reached the A-site. These binding locations were confirmed experimentally by measuring competitive inhibition constants K I2 for propidium and tacrine, inhibitors specific for the P-and A-sites, respectively. Values of K I2 for propidium increased 30-to 100-fold when ligands had either intermediate or long tethers. In contrast, the value of K I2 for tacrine increased substantially only when ligands had long tethers. These relative changes in propidium and tacrine affinities thus provided a sensitive molecular ruler for assigning the binding locations of the tethered cations.The primary physiological role of acetylcholinesterase (AChE) 1 is to hydrolyze the neurotransmitter acetylcholine at cholinergic synapses. The AChE structure has evolved to carry out this hydrolysis at rates that are among the highest known for enzyme-catalyzed reactions (1). One feature of the AChE catalytic pathway is the formation of an intermediate acyl enzyme that is hydrolyzed by water. It has long been known that AChE forms an acyl enzyme not only with carboxyl esters like acetylcholine but also with carbamic acid esters and phosphoric acid esters (also called organophosphates or OPs) and that these intermediates differ dramatically in their deacylation rate constants (2-5). In particular, organophosphorylated AChEs are hydrolyzed some 10 10 times slower than acetylated AChE and are effectively inactivated. OP inactivation of AChE results in failure of cholinergic synaptic transmission, deterioration of neuromuscular junctions, flaccid muscle paralysis, an...

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[13] Acetylcholinesterase

Cited by 34 publications

References 24 publications

A small hydrophobic domain that localizes human erythrocyte acetylcholinesterase in liposomal membranes is cleaved by papain digestion

A small hydrophobic domain that localizes human erythrocyte acetylcholinesterase in liposomal membranes is cleaved by papain digestion

Ultrastructure and composition of bovine conglutinin

Inhibitors Tethered Near the Acetylcholinesterase Active Site Serve as Molecular Rulers of the Peripheral and Acylation Sites

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