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Biomedical subjects

I Silman

Publications and source records attributed to I Silman.

At least 19 recordsLinked to original sources
PubMed1992

Chemical modification of Torpedo acetylcholinesterase by disulfides: appearance of a "molten globule" state.

Modification of Torpedo californica acetylcholinesterase (AChE) both by bis(1-oxy-2,2,5,5-tetramethyl-3-imidazolin-4-yl)disulfide (biradical) and by 4,4'-dithiopyridine, via a thiol-disulfide exchange reaction, was monitored by EPR and optical spectroscopy, respectively. Incubation with these reagents caused complete loss of enzymic activity. Treatment with glutathione of AChE modified by either of the two disulfides led to rapid release of the bound reagent with simultaneous regeneration of the single free thiol group of the enzyme. However, no concomitant recovery of catalytic activity was observed. SDS-PAGE showed that both the modified and demodified enzymes retained their structure as a disulfide-linked dimer. Circular dichroism revealed that modification of AChE by the disulfide agents with or without demodification by glutathione led to a complete disappearance of the ellipticity in the near-UV and to a much smaller decrease in ellipticity in the far-UV. The CD spectra observed are typical of the "molten globule" state of proteins. 1-Anilino-8-naphthalenesulfonate binding measurements and an enhanced susceptibility to trypsinolysis supported the supposition that chemical modification had transformed native AChE to a "molten globule".

Acetylcholinesterase↗
PubMed1992

Conversion of acetylcholinesterase to butyrylcholinesterase: modeling and mutagenesis.

Torpedo acetylcholinesterase (AcChoEase, EC 3.1.1.7) and human butyrylcholinesterase (BtChoEase, EC 3.1.1.8), while clearly differing in substrate specificity and sensitivity to inhibitors, possess 53% sequence homology; this permitted modeling human BtChoEase on the basis of the three-dimensional structure of Torpedo AcChoEase. The modeled BtChoEase structure closely resembled that of AcChoEase in overall features. However, six conserved aromatic residues that line the active-site gorge, which is a prominent feature of the AcChoEase structure, are absent in BtChoEase. Modeling showed that two such residues, Phe-288 and Phe-290, replaced by leucine and valine, respectively, in BtChoEase, may prevent entrance of butyrylcholine into the acyl-binding pocket. Their mutation to leucine and valine in AcChoEase, by site-directed mutagenesis, produced a double mutant that hydrolyzed butyrylthiocholine almost as well as acetylthiocholine. The mutated enzyme was also inhibited well by the bulky, BtChoEase-selective organophosphate inhibitor (tetraisopropylpyrophosphoramide, iso-OMPA). Trp-279, at the entrance of the active-site gorge in AcChoEase, is absent in BtChoEase. Modeling designated it as part of the "peripheral" anionic site, which is lacking in BtChoEase. The mutant W279A displayed strongly reduced inhibition by the peripheral site-specific ligand propidium relative to wild-type Torpedo AcChoEase, whereas inhibition by the catalytic-site inhibitor edrophonium was unaffected.

Acetylcholinesterase↗
PubMed1992

Site-directed mutagenesis of active-site-related residues in Torpedo acetylcholinesterase. Presence of a glutamic acid in the catalytic triad.

Site-directed mutagenesis was used to investigate the role of acidic amino acid residues close to the active site of Torpedo acetylcholinesterase. The recently determined atomic structure of this enzyme shows the conserved Glu-327, together with His-440 and Ser-200 as forming a catalytic triad, while the adjacent conserved Asp-326 points away from the active site. Transfection of appropriately mutated DNA into COS cells showed that the mutation of Asp-326----Asn had little effect on catalytic activity or the molecular forms expressed, suggesting no crucial structural or functional role for this residue. Mutation of Glu-327 to Gln or to Asp led to an inactive product. These results support the conclusions of the structural analysis for the two acidic residues.

Acetylcholinesterase↗
PubMed1992

G2-acetylcholinesterase is presynaptically localized in Torpedo electric organ.

In Torpedo electric organ, much of the acetylcholinesterase (AChE) is a globular dimer (G2), anchored to the plasma membrane via covalently attached phosphatidylinositol and selectively solubilized by a bacterial phosphatidylinositol-specific phospholipase C. While the structure of this form of the enzyme is well-established, the ultrastructural localization of G2-AChE is still unclear. Selective solubilization with phosphatidylinositol-specific phospholipase C was, therefore, combined with immunocytochemistry at the electron microscope level, in order to localize G2-AChE in electric organ of Torpedo ocellata. Thin sections of electric organ were labelled with antibodies raised against Torpedo AChE, followed by gold-conjugated second antibodies, before or after exposure to the phospholipase. For comparison, the location of AChE was examined using histochemical methods. We show that (1) immunolabelling is concentrated in the synaptic clefts between nerve terminals and the innervated face of the electrocyte; (2) this labelling co-localizes with AChE histochemical reaction products; and (3) prior exposure to the phospholipase causes a decrease in AChE-associated labelling. Quantitative analysis of immunolabelling in the synaptic clefts shows that the phospholipase treatment had reduced primary labelling at or adjacent to the presynaptic membrane. Together with our earlier biochemical and immunofluorescent evidence, these results support our previous assignment of a neuronal and synaptic localization for G2-AChE in Torpedo electric organ.

Acetylcholinesterase↗
PubMed1992

The alpha/beta hydrolase fold.

We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.

Acetylcholinesterase↗
PubMed1991

Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein.

The three-dimensional structure of acetylcholinesterase from Torpedo californica electric organ has been determined by x-ray analysis to 2.8 angstrom resolution. The form crystallized is the glycolipid-anchored homodimer that was purified subsequent to solubilization with a bacterial phosphatidylinositol-specific phospholipase C. The enzyme monomer is an alpha/beta protein that contains 537 amino acids. It consists of a 12-stranded mixed beta sheet surrounded by 14 alpha helices and bears a striking resemblance to several hydrolase structures including dienelactone hydrolase, serine carboxypeptidase-II, three neutral lipases, and haloalkane dehalogenase. The active site is unusual because it contains Glu, not Asp, in the Ser-His-acid catalytic triad and because the relation of the triad to the rest of the protein approximates a mirror image of that seen in the serine proteases. Furthermore, the active site lies near the bottom of a deep and narrow gorge that reaches halfway into the protein. Modeling of acetylcholine binding to the enzyme suggests that the quaternary ammonium ion is bound not to a negatively charged "anionic" site, but rather to some of the 14 aromatic residues that line the gorge.

Acetylcholine↗
PubMed1979

Immunological cross-reactivity between electric-eel acetylcholinesterase and rat-tail-tendon collagen.

Immunological cross-reactivity between acetylcholinesterase from the electric organ of the electric eel and rat tail tendon collagen was examined both on the cellular and humoral levels. 1. Guinea pigs immunized with rat tail tendon collagen displayed a strong delayed-type skin reaction when tested with the elongated acetylcholinesterase preparation (i.e. 14-S + 18-S molecular forms). However, when the glubular 11-S enzyme was tested, almost no cross-reactivity was obtained. Similarly, guinea pigs immunized with 14-S + 18-S preparation exhibited skin sensitization to rat tail tendon collagen. 2. Using a radioimmunoassay, it was observed that 125I-labeled 14-S + 18-S acetylcholinesterase binds efficiently to rabbit antiserum elicited against rat tail tendon collagen, whereas 125I-labeled 11-S enzyme does not bind at all to this antiserum. Similar results were obtained by passive hemagglutination assay. The experiments suggest that 14-S + 18-S acetylcholinesterase, but not 11-S enzyme, which is devoid of the tail structure, has antigenic determinants in common with collagen from rat tail tendon.

Acetylcholinesterase↗
PubMed1977

Effects of quaternary ligands on the inhibition of acetylcholinesterase by arsenite.

Arsenite inhibits acetylcholinesterase in a second-order reaction. The rate and equilibrium constants depend upon pH and have values on the order of 10(2) M-1 min-1 and 10(5) M (dissociation), respectively. Some quaternary ammonium ligands completely block the arsenite inhibition of the enzyme, others decrease the rate of the reaction and some, notably pyridine-2 aldoxime methiodide, greatly accelerate the rate of the reaction, up to 220-fold. Accelerators may bind at a separate enzyme site distinct form the anionic site involved in substrate binding. Although the kinetic data are consistent with a covalent reaction between arsenite and acetylcholinesterase, chemical evidence excludes the involvement of sulfhydryl groups which are usually implicated in arsenite inhibition.

Ammonia↗