torpedo ink

Recent studies have demonstrated several biological activities of curcumin with therapeutic potential against Alzheimer's disease, among them the inhibition of the enzyme acetylcholinesterase (AChE). Aiming at identifying the chemical features relevant for this activity, the inhibition of curcumin and a set of 7 derivatives against AChE of E. electricus was measured. These derivatives presented lower activity than curcumin, allowing for the identification of possible unfavorable enzyme-inhibitor interactions. Our computational approach was to dock the molecules to the active site of AChE, followed by an analysis of hydrogen bonds and close contacts to relevant aromatic amino acid residues. To account for inhibitory activity, we sought to define the common structural features between known acetylcholinesterase inhibitors and the tested derivatives. A pharmacophore model was generated, which consisted of two hydrophobic, one aromatic and one hydrogen bond acceptor features. We conclude that the presence of two aromatic rings and the distance between them, allows curcumin and its derivatives to favorably interact with both the quaternary and peripheral sites of AChE. Hydrogen bonds can be formed with the quaternary and acyl sites, which should further stabilize the complex. The acylation of the hydroxyl groups and the reduction of the conjugated double bonds lowered the inhibitory activity, pointing to the modification of the keto-enol moiety as the best alternative for the design of more potent curcumin derivatives as acetylcholinesterase inhibitors.

The presynaptic plasma membrane (PSPM) of cholinergic nerve terminals was purified from Torpedo electric organ using a large-scale procedure. Up to 500 g of frozen electric organ were fractioned in a single run, leading to the isolation of greater than 100 mg of PSPM proteins. The purity of the fraction is similar to that of the synaptosomal plasma membrane obtained after subfractionation of Torpedo synaptosomes as judged by its membrane-bound acetylcholinesterase activity, the number of Glycera convoluta neurotoxin binding sites, and the binding of two monoclonal antibodies directed against PSPM. The specificity of these antibodies for the PSPM is demonstrated by immunofluorescence microscopy.

Structure-based drug design utilizes apo-protein or complex structures retrieved from the PDB. >57% of crystallographic PDB entries were obtained with polyethyleneglycols (PEGs) as precipitant and/or as cryoprotectant, but >6% of these report presence of individual ethyleneglycol oligomers. We report a case in which ethyleneglycol oligomers' presence in a crystal structure markedly affected the bound ligand's position. Specifically, we compared the positions of methylene blue and decamethonium in acetylcholinesterase complexes obtained using isomorphous crystals precipitated with PEG200 or ammonium sulfate. The ligands' positions within the active-site gorge in complexes obtained using PEG200 are influenced by presence of ethyleneglycol oligomers in both cases bound to W84 at the gorge's bottom, preventing interaction of the ligand's proximal quaternary group with its indole. Consequently, both ligands are ∼3.0Å further up the gorge than in complexes obtain...

The interaction of general anesthetics at the lipid/protein interface of the nicotinic acetylcholine receptor reconstituted in dioleoylphosphatidylcholine bilayers at various lipid/protein ratios has been studied using the electron spin resonance spectra of phosphatidylcholine spin-labeled at the fourtheenth acyl carbon (14-PCSL). In addition to the bilayer spectrum, the spin label reported a more motionally restricted environment whose contribution increased with increasing protein/lipid ratio. Exchange between thses ...

Barbiturate-induced anesthesia is a complex mechanism that probably involves several ligand-gated ion channel superfamilies. One of these superfamilies includes the archetypical nicotinic acetylcholine receptor (nAChR), in which barbiturates act as noncompetitive antagonists. In this regard, we used the Torpedo californica nAChR and a series of barbiturate analogs to characterize the barbiturate binding site(s) on this superfamily member. [(14)C]Amobarbital binds to one high-affinity (K(d) = 3.7 microM) and several (approximately 11) low-affinity (K(d) = 930 microM) sites on the resting and desensitized nAChRs, respectively. Characteristics of the barbiturate binding site on the resting nAChR include: (1) a tight structure-activity relationship. For example, the barbiturate isobarbital [5-ethyl-5'-(2-methylbutyl) barbituric acid] is >10-fold less potent than its formula isomer amobarbital [5-ethyl-5'-(3-methylbutyl) barbituric acid] in inhibiting [(14)C]amobarbital bindin...

Pharmacological properties of the (+)- and (-)-isomers of synthetic epibatidine, exo-2-(6-chloro-3-pyridyl)-7-azabicyclo-[2.2.1]heptane, were compared with nicotine and acetylcholine on several subtypes of chicken and human nicotinic acetylcholine receptors (AChRs). Both isomers of epibatidine behaved as extremely potent full agonists on chicken (alpha 3 beta 2, alpha 3 beta 4, alpha 4 beta 2, alpha 7, and alpha 8) and human (alpha 3 beta 2, alpha 3 beta 4, and alpha 7) neuronal AChRs expressed in Xenopus oocytes. Currents induced by epibatidine were effectively blocked by the nicotinic antagonists hexamethonium and mecamylamine. Apparent affinity was 100 to 1000-fold higher for epibatidine than for nicotine or acetylcholine. EC50 values ranged from 1 nM (for homomeric chicken alpha 8) to 2 microM (for homomeric chicken alpha 7). Epibatidine showed comparatively lower affinity for muscle-type AChRs from Torpedo and humans (EC50 values, 1.6 and 16 microM respectively). In binding ass...

We assessed the pharmacological activity of anabaseine, a toxin found in certain animal venoms, relative to nicotine and anabasine on a variety of vertebrate nicotinic receptors, using cultured cells, the Xenopus oocyte expression system, contractility assays with skeletal and smooth muscle strips containing nicotinic receptors and in vivo rat prostration assay involving direct injection into the lateral ventricle of the brain. Anabaseine stimulated every subtype of nicotinic receptor that was tested. It was the most potent frog skeletal muscle nicotinic receptor agonist. At higher concentrations it also blocked the BC3H1 (adult mouse) muscle type receptor ion channel. The affinities of the three nicotinoid compounds for rat brain membrane alpha-bungarotoxin binding sites and their potencies for stimulating Xenopus oocyte homomeric alpha7 receptors, expressed in terms of their active monocation concentrations, displayed the same rank order, anabaseine>anabasine> nicotine. Alth...

Recently there has been increased interest in pancreatic cholesterol esterase due to correlation between enzymatic activity in vivo and absorption of dietary cholesterol. Cholesterol esterase plays a role in digestive lipid absorption in the upper intestinal tract, though its role in cholesterol absorption in particular is controversial. Serine lipases, acetylcholinesterase, butyrylcholinesterase, and cholesterol esterase belong to a large family of proteins called the alpha/beta-hydrolase fold, and they share the same catalytic machinery as serine proteases in that they have an active site serine residue which, with a histidine and an aspartic or glutamic acid, forms a catalytic triad. The aim of this work is to study the stereoselectivity of the acyl chain binding site of the enzyme for four diastereomers of an inhibitor. Four diastereomers of 2'-N-alpha-methylbenzylcarbamyl-1, 1'-bi-2-naphthol (1) are synthesized from the condensation of R-(+)- or S-(-)-1, 1'-bi-2-nap...

Various acetylcholine receptor-rich membrane preparations from Torpedo californica electroplax tissue were examined using the techniques of differential scanning calorimetry coupled with gel electrophoretic analysis of heat-denaturing material and functional assays following passage through discrete transitions. In unfractionated membranes, four irreversible calorimetric transitions were observed, one of which (Td = 59 degrees C) could be assigned to a complete loss of acetylcholine receptor function. A second lower temperature transition apparently corresponds to loss of certain peripheral membrane proteins including the Mr = 43,000 polypeptide and the acetylcholinesterase activity. Membrane preparations highly enriched in acetylcholine receptor polypeptides contained a major transition at 59 degrees C which could be shown to be sensitive to the presence of added ligands of the acetylcholine receptor, supporting its assignment to structural alterations of the receptor protein or it...

Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a su...

An alpha-bungarotoxin-sensitive nicotinic cholinergic receptor from chick optic lobe has been completely purified. Its standard sedimentation coefficient is 9.1 S. The value near 12 S reported for the related component from other brain regions can be reproduced when ...