Glycosylated-imidazole aldoximes as reactivators of pesticides inhibited AChE: Synthesis and in-vitro reactivation study.

The present armamentarium of commercially available antidotes provides limited protection against the neurological effects of organophosphate exposure. Hence, there is an urgent need to design and develop molecules that can protect and reactivate inhibited-AChE in the central nervous system. Some natural compounds like glucose and certain amino acids (glutamate, the anion of glutamic acid) can easily cross the blood brain barrier although they are highly polar. Glucose is mainly transported by systems like glucose transporter protein type 1 (GLUT1). For this reason, a series of non-quaternary and quaternary glycosylated imidazolium oximes with different alkane linkers have been designed and synthesized. These compounds were evaluated for their in-vitro reactivation ability against pesticide (paraoxon-ethyl and paraoxon-methyl) inhibited-AChE and compared with standards antidote AChE reactivators pralidoxime and obidoxime. Several physicochemical properties including acid dissociation constant (pKa), logP, logD, HBD and HBA, have also been assessed for reported compounds. Out of the synthesized compounds, three have exhibited comparable potency with a standard antidote (pralidoxime).

Kinetics of acetylcholinesterase inhibition by hemp seed protein-derived peptides.

The aim of this work was to enhance the acetylcholinesterase (AChE)-inhibitory activity of a pepsin-produced hemp seed protein hydrolysates (HPH) through reverse-phase HPLC separation followed by identification of peptide sequences present in the most active fraction. The HPH was separated into eight fractions (F1-F8) with F7 exhibiting significantly (p < 0.05) the strongest (97.5%) in vitro inhibition of electric eel AChE (eeAChE) activity in comparison to 53.8% for HPH. The HPH consisted mostly of low molecular weight peptides of < 11 amino acid residues and most contained at least one hydrophobic amino acid. Kinetics of enzyme inhibition revealed a mixed-type inhibition of eeAChE activity by HPH whereas F7 acted through an uncompetitive mode; in contrast inhibition of human AChE by HPH and F7 was uncompetitive. The stronger inhibitory potency of the F7 peptides fraction against both enzymes was confirmed through reduced maximal velocity, catalytic efficiency, and inhibition constant values when compared to the HPH. PRACTICAL APPLICATIONS: The use of natural products for the prevention or treatment of human diseases continues to be an area of intense research activities. Food protein-derived peptides obtained through enzymatic hydrolysis of hemp seed proteins were shown in vitro to be strong inhibitors of activities of both the eel and human forms of acetylcholinesterase (AChE). AChE is an important therapeutic target because excessive activity of this enzyme is a causative factor of neurodegenerative diseases such as dementia and Alzheimer's. This work showed that peptides in the most active fraction are small in sizes, which may favor their absorption into blood circulation and possible permeation of the blood-brain barrier. Therefore, the hemp seed peptides are potential agents that can be used to formulate functional foods and nutraceuticals against neurodegenerative diseases.

Chemical profile of Lippia thymoides, evaluation of the acetylcholinesterase inhibitory activity of its essential oil, and molecular docking and molecular dynamics simulations.

The essential oils of the fresh and dry flowers, leaves, branches, and roots of Lippia thymoides were obtained by hydrodistillation and analyzed using gas chromatography (GC) and GC-mass spectrometry (MS). The acetylcholinesterase inhibitory activity of the essential oil of fresh leaves was investigated on silica gel plates. The interactions of the key compounds with acetylcholinesterase were simulated by molecular docking and molecular dynamics studies. In total, 75 compounds were identified, and oxygenated monoterpenes were the dominant components of all the plant parts, ranging from 19.48% to 84.99%. In the roots, the main compounds were saturated and unsaturated fatty acids, having contents varying from 39.5% to 32.17%, respectively. In the evaluation of the anticholinesterase activity, the essential oils (detection limit (DL) = 0.1 ng/spot) were found to be about ten times less active than that of physostigmine (DL = 0.01ng/spot), whereas thymol and thymol acetate presented DL values each of 0.01 ng/spot, equivalent to that of the positive control. Based on the docking and molecular dynamics studies, thymol and thymol acetate interact with the catalytic residues Ser203 and His447 of the active site of acetylcholinesterase. The binding free energies (ΔGbind) for these ligands were -18.49 and -26.88 kcal/mol, demonstrating that the ligands are able to interact with the protein and inhibit their catalytic activity.

Acetamide Derivatives of Chromen-2-ones as Potent Cholinesterase Inhibitors.

Alzheimer's disease (AD), a neurodegenerative disorder, is a serious medical issue worldwide with drastic social consequences. Inhibition of cholinesterase is one of the rational and effective approaches to retard the symptoms of AD and, hence, consistent efforts are being made to develop efficient anti-cholinesterase agents. In pursuit of this, a series of 19 acetamide derivatives of chromen-2-ones were synthesized and evaluated for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory potential. All the synthesized compounds exhibited significant anti-AChE and anti-BChE activity, with IC50 values in the range of 0.24-10.19 µM and 0.64-30.08 µM, respectively, using donepezil hydrochloride as the standard. Out of 19 compounds screened, 3 compounds, viz. 22, 40, and 43, caused 50% inhibition of AChE at 0.24, 0.25, and 0.25 µM, respectively. A kinetic study revealed them to be mixed-type inhibitors, binding with both the CAS and PAS sites of AChE. The above-selected compounds were found to be effective inhibitors of AChE-induced and self-mediated Aß1-42 aggregation. ADMET predictions demonstrated that these compounds may possess suitable blood-brain barrier (BBB) permeability. Hemolytic assay results revealed that these compounds did not lyse human RBCs up to a thousand times of their IC50 value. MTT assays performed for the shortlisted compounds showed them to be negligibly toxic after 24 h of treatment with the SH-SY5Y neuroblastoma cells. These results provide insights for further optimization of the scaffolds for designing the next generation of compounds as lead cholinesterase inhibitors.

Screen-printed electrode modified with carbon black and chitosan: a novel platform for acetylcholinesterase biosensor development.

We report a screen-printed electrode (SPE) modified with a dispersion of carbon black (CB) and chitosan by drop casting. A cyclic voltammetry technique towards ferricyanide, caffeic acid, hydroquinone, and thiocholine was performed and an improvement of the electrochemical response with respect to bare SPE as well as SPE modified only with chitosan was observed. The possibility to detect thiocholine at a low applied potential with high sensitivity was exploited and an acetylcholinesterase (AChE) biosensor was developed. A dispersion of CB, chitosan, and AChE was used to fabricate this biosensor in one step by drop casting. The enzymatic activity of the immobilized AChE was determined measuring the enzymatic product thiocholine at +300 mV. Owing to the capability of organophosphorus pesticides to inhibit AChE, this biosensor was used to detect these pollutants, and paraoxon was taken as model compound. The enzyme inhibition was linearly related to the concentration of paraoxon up to 0.5 µg L(-1), and a low detection limit equal to 0.05 µg L(-1) (calculated as 10% of inhibition) was achieved. This biosensor was challenged for paraoxon detection in drinking waters with satisfactory recovery values. The use of AChE embedded in a dispersion of CB and chitosan allowed an easy and fast production of a sensitive biosensor suitable for paraoxon detection in drinking waters at legal limit levels. Graphical Abstract Biosensors based on screen-printed electrodes modified with Acetylcholinesterase, Carbon Black, and Chitosan for organophosphorus pesticide detection.

Converting maslinic acid into an effective inhibitor of acylcholinesterases.

During the last decade, maslinic acid has been evaluated for many biological properties, e.g. as an anti-tumor or an anti-viral agent but also as a nutraceutical. The potential of maslinic acid and related derivatives to act as inhibitors of acetyl- or butyryl-cholinesterase was examined in this communication in more detail. Cholinesterases do still represent an interesting group of target enzymes with respect to the investigation and treatment of the Alzheimer's disease and other dementia illnesses as well. Although other triterpenoic acids have successfully been tested for their ability to act as inhibitors of cholinesterases, up to now maslinic acid has not been part of such studies. For this reason, three series of maslinic acid derivatives possessing modifications at different centers were synthesized and subjected to Ellman's assay to determine their inhibitory strength and type of inhibitory action. While parent compound maslinic acid was no inhibitor in these assays, some of the compounds exhibited an inhibition of acetylcholinesterase in the single-digit micro-molar range. Two compounds were identified as inhibitors of butyrylcholinesterase showing inhibition constants comparable to those of galantamine, a drug often used in the treatment of Alzheimer's disease. Furthermore, additional selectivity as well as cytotoxicity studies were performed underlining the potential of several derivatives and qualifying them for further investigations. Docking studies revealed that the different kinetic behavior within the same compound series may be explained by the ability of the compounds to enter the active site gorge of AChE.

Na+/K+-ATPase α-subunit (nkaα) isoforms and their mRNA expression levels, overall Nkaα protein abundance, and kinetic properties of Nka in the skeletal muscle and three electric organs of the electric eel, Electrophorus electricus.

This study aimed to obtain the coding cDNA sequences of Na+/K+-ATPase α (nkaα) isoforms from, and to quantify their mRNA expression in, the skeletal muscle (SM), the main electric organ (EO), the Hunter's EO and the Sach's EO of the electric eel, Electrophorus electricus. Four nkaα isoforms (nkaα1c1, nkaα1c2, nkaα2 and nkaα3) were obtained from the SM and the EOs of E. electricus. Based on mRNA expression levels, the major nkaα expressed in the SM and the three EOs of juvenile and adult E. electricus were nkaα1c1 and nkaα2, respectively. Molecular characterization of the deduced Nkaα1c1 and Nkaα2 sequences indicates that they probably have different affinities to Na+ and K+. Western blotting demonstrated that the protein abundance of Nkaα was barely detectable in the SM, but strongly detected in the main and Hunter's EOs and weakly in the Sach's EO of juvenile and adult E. electricus. These results corroborate the fact that the main EO and Hunter's EO have high densities of Na+ channels and produce high voltage discharges while the Sach's EO produces low voltage discharges. More importantly, there were significant differences in kinetic properties of Nka among the three EOs of juvenile E. electricus. The highest and lowest Vmax of Nka were detected in the main EO and the Sach's EO, respectively, with the Hunter's EO having a Vmax value intermediate between the two, indicating that the metabolic costs of EO discharge could be the highest in the main EO. Furthermore, the Nka from the main EO had the lowest Km (or highest affinity) for Na+ and K+ among the three EOs, suggesting that the Nka of the main EO was more effective than those of the other two EOs in maintaining intracellular Na+ and K+ homeostasis and in clearing extracellular K+ after EO discharge.

Biosensor based on acetylcholinesterase immobilized onto layered double hydroxides for flow injection/amperometric detection of organophosphate pesticides.

We developed a highly sensitive flow injection/amperometric biosensor for the detection of organophosphate pesticides (OPs) using layered double hydroxides (LDHs) as the immobilization matrix of acetylcholinesterase (AChE). LDHs provided a biocompatible microenvironment to keep the bioactivity of AChE, due to the intrinsic properties of LDHs (such as a regular structure, good mechanical, chemical and thermal stabilities, and swelling properties). By integrating the flow injection analysis (FIA) with amperometric detection, the resulting AChE-LDHs modified electrode greatly catalyzed the oxidation of the enzymatically generated thiocholine product, and facilitated the detection automation, thus increasing the detection sensitivity. The analytical conditions for the FIA/amperometric detection of OPs were optimized by using methyl parathion (MP) as a model. The inhibition of MP was proportional to its concentration ranging from 0.005 to 0.3µg mL(-1) and 0.3 to 4.0µg mL(-1) with a detection limit 0.6ng mL(-1) (S/N=3). The developed biosensor exhibited good reproducibility and acceptable stability.

Exploration of two-enzyme coupled catalysis system using scanning electrochemical microscopy.

In biological metabolism, a given metabolic process usually occurs via a group of enzymes working together in sequential pathways. To explore the metabolism mechanism requires the understanding of the multienzyme coupled catalysis systems. In this paper, an approach has been proposed to study the kinetics of a two-enzyme coupled reaction using SECM combining numerical simulations. Acetylcholine esterase and choline oxidase are immobilized on cysteamine self-assembled monolayers on tip and substrate gold electrodes of SECM via electrostatic interactions, respectively. The reaction kinetics of this two-enzyme coupled system upon various separation distance precisely regulated by SECM are measured. An overall apparent Michaelis-Menten constant of this enzyme cascade is thus measured as 2.97 mM at an optimal tip-substrate gap distance of 18 µm. Then, a kinetic model of this enzyme cascade is established for evaluating the kinetic parameters of individual enzyme by using the finite element method. The simulated results demonstrate the choline oxidase catalytic reaction is the rate determining step of this enzyme cascade. The Michaelis-Menten constant of acetylcholine esterase is evaluated as 1.8 mM. This study offers a promising approach to exploring mechanism of other two-enzyme coupled reactions in biological system and would promote the development of biosensors and enzyme-based logic systems.

An in vitro perspective to cholinesterase inhibitory and antioxidant activity of five Gentiana species and Gentianella caucasea.

The dichloromethane (DCM), ethyl acetate (EtOAc), and methanol extracts from the leaves, roots, and flowers of the five species of Gentiana (Gentiana asclepiadea, Gentiana cruciata, Gentiana olivieri, Gentiana septemfida, and Gentiana verna) and Gentianella caucasea were investigated for their inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and antioxidant effect using 2,2-diphenyl-1-picrylhydrazyl radical scavenging, metal-chelation capacity, and ferric-reducing antioxidant power assays. Total phenol and flavonoid contents of the extracts were determined spectrophotometrically. The presence of some characteristic compounds found in Gentiana species (gentiopicroside, swertiamarin, isoorientin, isovitexin and vitexin) was analyzed in the extracts by thin layer chromatography. The flower DCM extract of G. verna exerted the highest inhibition against AChE (53.65 ± 1.03%), whereas the root EtOAc extract of G. cruciata was the most effective in BChE inhibition assay (50.72 ± 0.75%) at 100 µg ml⁻¹. The extracts of G. verna were also found to be more active in the antioxidant tests.

Acetylcholine esterase inhibitors in effluents from oil production platforms in the North Sea.

Inhibition of acetylcholine esterase (AChE) activity is a biomarker for the exposure to neurotoxic compounds such as organophosphates and is intimately associated with the toxicity of several pesticides. In the present study, the AChE inhibiting potential of organic extracts of production water (produced water) from oil and gas production platforms in the Norwegian sector of the North Sea was determined in an in vitro bioassay based on commercially available purified AChE from the electric organ of Electrophorus electricus (L.). The results from the studies show that produced water contains a combination of AChE inhibiting compounds and compounds stimulating AChE enzymatic activity. The AChE inhibition was predominantly caused by unidentified aromatic compounds in the oil/particulate fraction of produced water, whereas polar compounds in both the water soluble and oil/particulate fraction of produced water caused an apparent stimulation of AChE activity. Substrate saturation studies with fixed concentrations of produced water extracts confirmed that the inhibition occurred in a non-destructive and competitive manner. The concentrations of AChE inhibitors (7.9-453 ng paraoxon-equivalents L⁻¹, 2.2-178 µg dichlorvos-equivalents L⁻¹) were in many cases found to be several orders of magnitude higher than background levels. The findings demonstrate that produced water contains potentially neurotoxic compounds and suggest that further laboratory studies with fish or field studies in the vicinity of oil production facilities are highly warranted.

Novel piperazine derivative PMS1339 exhibits tri-functional properties and cognitive improvement in mice.

Amyloid-beta-induced neuroinflammation plays a central role in the extensive loss of cholinergic neurons and cognitive decline in Alzheimer's disease. The acetylcholinesterase (AChE) inhibitors are the first class of drugs used to enhance surviving cholinergic activities. However, their limited effectiveness following long-term treatment raises a need for new multi-target therapies. We report herein a novel piperazine derivative compound PMS1339 possesses multifunctional properties including anti-platelet-activating factor, AChE inhibition, Abeta aggregation inhibition and cognitive improvement. PMS1339 could significantly inhibit both mice brain AChE (IC50=4.41+/-0.63 microM) and sera butyrylcholinesterase (BuChE, IC50=1.09+/-0.20 microM). PMS1339 was also found to inhibit neuronal AChE secreted by SH-SY5Y cell line (IC50=17.95+/-2.31 microM). Enzyme kinetics experiments performed on electric eel AChE indicated that PMS1339 acts as a mixed type competitive AChE inhibitor. Molecular docking studies using the X-ray crystal structure of AChE from Torpedo californica elucidated the interactions between PMS1339 and AChE: PMS1339 is well buried inside the active-site gorge of AChE interacting with Trp84 at the bottom, Tyr121 halfway down and Trp279 at the peripheral anionic site (PAS). Thioflavin T-based fluorimetric assay revealed the ability of PMS1339 to inhibit AChE-induced Abeta aggregation. In-vivo study indicated PMS1339 (1 mg/kg i.p.) reversed scopolamine-induced memory impairment in mice. Overall, these findings indicated that PMS1339 exhibits tri-functional properties in vitro and cognitive improvement in vivo, and revealed the emergence of a multi-target-directed ligand to tackle the determinants of Alzheimer's disease.

Benzene-1,2-, 1,3-, and 1,4-di-N-substituted carbamates as conformationally constrained inhibitors of acetylcholinesterase.

Benzene-1,2-, 1,3-, and 1,4-di-N-substituted carbamates (1-15) are synthesized as the conformationally constrained inhibitors of acetylcholinesterase and mimic gauche, eclipsed, and anti-conformations of acetylcholine, respectively. All carbamates 1-15 are characterized as the pseudo substrate inhibitors of acetylcholinesterase. For a series of geometric isomers, the inhibitory potencies are as follows: benzene-1,4-di-N-substituted carbamate (para compound) > benzene-1,3-di-N-substituted carbamate (meta compound) > benzene-1,2-di-N-substituted carbamate (ortho compound). Therefore, benzene-1,4-di-N-substituted carbamates (para compounds), with the angle of 180 degrees between two C(benzene)-O bonds, mimic the preferable anti C-O/C-N conformers of acetylcholine for the choline ethylene backbone in the acetylcholinesterase catalysis.

Mechanisms of cholinesterase inhibition by inorganic mercury.

The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X-ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo-first-order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury-induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.

Ca2+/calmodulin-dependent protein kinase II is an essential mediator in the coordinated regulation of electrocyte Ca2+-ATPase by calmodulin and protein kinase A.

The aim of this study was to investigate (a) whether Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) participates in the regulation of plasma membrane Ca2+-ATPase and (b) its possible cross-talk with other kinase-mediated modulatory pathways of the pump. Using isolated innervated membranes of the electrocytes from Electrophorus electricus L., we found that stimulation of endogenous protein kinase A (PKA) strongly phosphorylated membrane-bound CaM kinase II with simultaneous substantial activation of the Ca2+ pump (approximately 2-fold). The addition of cAMP (5-50 pM), forskolin (10 nM), or cholera toxin (10 or 100 nM) stimulated both CaM kinase II phosphorylation and Ca2+-ATPase activity, whereas these activation processes were cancelled by an inhibitor of the PKA alpha-catalytic subunit. When CaM kinase II was blocked by its specific inhibitor KN-93, the Ca2+-ATPase activity decreased to the levels measured in the absence of calmodulin; the unusually high Ca2+ affinity dropped 2-fold; and the PKA-mediated stimulation of Ca2+-ATPase was no longer seen. Hydroxylamine-resistant phosphorylation of the Ca2+-ATPase strongly increased when the PKA pathway was activated, and this phosphorylation was suppressed by inhibition of CaM kinase II. We conclude that CaM kinase II is an intermediate in a complex regulatory network of the electrocyte Ca2+ pump, which also involves calmodulin and PKA.

Polarized distribution of Na+, K(+)-ATPase alpha-subunit isoforms in electrocyte membranes.

We have previously demonstrated that Na+, K(+)-ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the alpha subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of alpha isoforms (alpha1 and alpha2) in the electrocyte. Dose-response curves showed that non-innervated membranes present a Na+, K(+)-ATPase activity 2.6-fold more sensitive to ouabain (I50=1.0+/-0.1 microM) than the activity of innervated membranes (I50=2.6+/-0.2 microM). As depicted in [3H]ouabain binding experiments, when the [3H]ouabain-enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na+, K(+)-ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of alpha1 and alpha2 isoforms, restricted to the innervated and non-innervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na+, K(+)-ATPase alpha-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell.

Choline acetyltransferase detection in normal and denervated electrocyte from Electrophorus electricus (L.) using a confocal scanning optical microscopy analysis

Acetylcholine is the neurotransmitter responsible for the transmission of impulses from cholinergic neurons to cells of innervated tissues. Its biosynthesis is catalyzed by the enzyme Choline acetyltransferase that is considered to be a phenotypically specific marker for cholinergic system. It is well known that the regulation of Choline acetyltransferase activity under physiological and pathological conditions is important for development and neuronal activities of cholinergic functions. We observed the distribution of Choline acetyltransferase in sections from the normal and denervated main electric organ sections of Electrophorus electricus (L.) by immunofluorescence using a anti-Choline acetyltransferase antibody. The animals were submitted to a surgical procedure to remove about 20 nerves and after 30 and 60 days, they were sacrificed. After 30 days, the results from immunohistochemistry demonstrated an increase on the Choline acetyltransferase distribution at denervated tissue sections when compared with the sections from the normal contralateral organ. A very similar labeling was observed between normal and denervated tissue sections of the animals after 60 days. However, Choline acetyltransferase activity (nmolesACh/ min/ mg of protein) in extracts obtained from electrocyte microsomal preparation, estimated by Fonnun's method (Fonnun 1975), was 70 per cent lower in the denervated extracts.

Chemical ligation to obtain proteins comprising helices with individual amino acid sequences.

Development of the strategies for assembling multiple kinds of peptide segments would give new possibilities for the de novo design of functional proteins. We will introduce our approach for the selective assembly of helical peptide segments on a peptide template to give four-helix-bundle proteins comprising individual helices.

Comparison of the inhibitory effects of mercury and cadmium on the creatine kinase from Electrophorus electricus (L).

We have determined the effects of mercury and cadmium on the creatine kinase activity of the electric organ of Electrophorus electricus (L.) which catalyses the transphosphorylation reaction between phosphocreatine and magnesium adenosine-5'-di-phosphate and has essential sulfhydryl groups. The kinetic effects of these heavy metals, which have high affinity for sulfhydryl groups, on the creatine kinase activity were analysed with the three reaction components: phosphocreatine, adenosine-5'-di-phosphate and magnesium. The kinetic data were analysed with a non-linear regression program (Sigmaplot for Windows). Both metals inhibit creatine kinase activity in the micromolar range, mercury being a more potent inhibitor than cadmium. With phosphocreatine as substrate, mercury behaved as a mixed partial hyperbolic inhibitor, non-competitive inhibitor with adenosine-5'-di-phosphate, and with magnesium mercury behaved as a competitive inhibitor. Cadmium inhibition was shown to be of a classical competitive nature with respect to both substrates, phosphocreatine or adenosine-5'-di-phosphate, and non-competitive when magnesium was the variable in the reaction mixture. The results suggest that the binding site of mercury is at or near the phosphocreatine site, but it is not the same as adenosine-5'-di-phosphate, whereas cadmium competes with these substrates to bind at the same sulphydryl site.

Active-site peptides of acetylcholinesterase of Electrophorus electricus: labelling of His-440 by 1-bromo-[2-14C]pinacolone and Ser-200 by tritiated diisopropyl fluorophosphate.

To characterize the structure of the active site of acetylcholinesterase (AChE) from the electric organ of E. electricus, we identified sites of incorporation of two active-site affinity labels, [3H]diisopropyl fluorophosphate ([3H]DFP), and 1-bromo-2-[14C]pinacolone ([14C]BrPin). AChE was isolated, purified, inactivated and digested with trypsin, and peptides containing 3H or 14C were purified by reverse-phase HPLC and characterized by N-terminal sequence analysis. [3H]DFP, labelling Ser-200, was found in a single peptide, QVTIFGESAGAASVGMHLLSPDSR, 83% identical with the sequence from Thr-193 to Arg-216 deduced for AChE of T. californica, with Gln, Ala, Leu, and Asp in place of Thr-193, Gly-203, Ile-210 and Gly-214, respectively, and 87% identical with that from bovine and human brain AChEs. Inactivation by [14C]BrPin led to two radioactive peptides. One, ASNLVWPEWMGVIHGYEIEFVFGLPLEK, was 96% identical with that extending from Ala-427 to Lys-454 of T. californica. Release of 14C in cycle 14 established reaction of [14C]BrPin with active-site His-440, protected by 5-trimethylammonio-2-pentanone (TAP). The other peptide, LLXVTENIDDAER, 77% homologous with that of T. californica extending from Leu-531 to Arg-543, had label associated with the third cycle, not protected by TAP, corresponding to Asn-533. The slow inactivation of eel AChE by reaction of [14C]BrPin at His-440 contrasts with that of AChE from T. nobiliana, where it reacts rapidly with a free cysteine, Cys-231, not present in eel AChE. For both AChEs, inactivation by BrPin prevents subsequent reaction with [3H]DFP, and prior inactivation by DFP does not prevent reactions with [14C]BrPin.