The importance of an efficient extraction protocol for the use of fish muscle cholinesterases as biomarkers.

Esterase activity found in muscle extracts is useful to evaluate harmful effects of anticholinesterase pollutants. Yet, most procedures applied in the extraction of fish muscle esterases in order to investigate their activity as a biomarker of environmental exposure comprise the homogenization of muscle tissue in low-salt solutions, followed by centrifugation to separate the supernatant as the enzyme source. However, acetylcholinesterase (AChE), the main target in these monitoring efforts, is a membrane-bound protein and is only present in muscle extracts if homogenization is carried out using chaotropic high-salt solutions. In this context, four extraction procedures using muscle tissue from six fish species were evaluated in order to establish a reproducible and reliable AChE assay for the determination of this biomarker. Results indicate that over 80% of AChE activity might be lacking in low-salt supernatants, and that the highest activities are obtained after extraction with solutions containing either 1molL-1 NaCl or 1molL-1 NaCl plus 3% Triton X-100, preserving almost 100% esterase activity over acetylthiocholine as substrate after centrifugation. Thus, many studies in the literature suffer from theoretical flaws and report erroneous AChE activity, since typical muscle AChE activity, the end-point biomarker for anticholinesterase pollutants, may have not been consistently assayed.

[Inhibitory effect of benzimidazole derivatives on cholinesterases of animals in the presence of different substrates].

Specifically synthesized group of benzimidazole derivatives possessing varying degrees of delocalization of the positive charge in the cation group of the molecule has been studied in order to search for potential cholinergically active compounds and to study the role of the Coulomb interaction in cholinesterase catalysis. These compounds were reversible inhibitors of cholinesterase (ChE) of human erythrocytes, horse serum, brain of the frog Rana temporaria and visual ganglia of the Pacific squid Todarodes pacificus in the presence of acetylthiocholine iodide and propionylthiocholine iodide as substrates. The differences in the nature of reversible inhibitory effect were observed. The effect of the inhibitor structure and substrate nature, specific for each of the studied inhibitors, on the character of the process of reversible inhibition was found.

Purification and studies on characteristics of cholinesterases from Daphnia magna.

Due to their significant value in both economy and ecology, Daphnia had long been employed to investigate in vivo response of cholinesterase (ChE) in anticholinesterase exposures, whereas the type constitution and property of the enzyme remained unclear. A type of ChE was purified from Daphnia magna using a three-step procedure, i.e., Triton X-100 extraction, ammonium sulfate precipitation, and diethylaminoethyl (DEAE)-Sepharose™-Fast-Flow chromatography. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), molecular mass of the purified ChE was estimated to be 84 kDa. Based on substrate studies, the purified enzyme preferred butyrylthiocholine iodide (BTCh) [with maximum velocity (Vmax)/Michaelis constant (Km)=8.428 L/(min·mg protein)] to acetylthiocholine iodide (ATCh) [with Vmax/Km=5.346 L/(min·mg protein)] as its substrate. Activity of the purified enzyme was suppressed by high concentrations of either ATCh or BTCh. Inhibitor studies showed that the purified enzyme was more sensitive towards inhibition by tetraisopropylpyrophosphoramide (iso-OMPA) than by 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284C51). Result of the study suggested that the purified ChE was more like a type of pseudocholinesterase, and it also suggested that Daphnia magna contained multiple types of ChE in their bodies.

ChE, GST and CAT: evaluation of the efficiency of a combined buffer for protein extraction.

Toxicity and biotransformation of several earthworm contaminants are widely evaluated nowadays using biochemical biomarkers. Many investigations track enzyme activities as biomarkers of neurotoxicity (cholinesterase (ChE)), metabolisation (glutathione-S-transferase (GST)) and oxidative stress (catalase (CAT)). This study proposes an evaluation of the use of a combined buffer, to extract proteins from earthworms and then analyse the 3 biomarkers. The method provides good results and allows protein extraction and quantitative determination of biomarkers with the same efficiency as the enzyme-specific buffers. It decreases preparation time and permits a study of the biomarkers on the same individual with only one homogenisation.

Cholinesterases from the marine mussels Mytilus galloprovincialis Lmk. and M. edulis L. from the freshwater bivalve Corbicula fluminea Müller.

In order to improve the molecular basis for the use of bivalve cholinesterases as a reliable biomarker for aquatic pollution, the polymorphism and characterization of these enzymes in Mytilus edulis, Mytilus galloprovincialis and Corbicula fluminea were investigated. All results are consistent with the presence of only one pharmacological form of cholinesterase in each species. The molecular masses were 180 kDa for the two marine mussels and 240 kDa for C. fluminea. The cholinesterases are anchored to the membrane by a glycosyl inositol phosphate like the Ga form (type I) described in vertebrates. Surprisingly, these cholinesterases were poorly inhibited by organophosphorous compounds compared to enzymes from other sources. This suggests that these bivalves could be used as a biomarker for acute rather than chronic contaminations by anticholinesterase insecticides.

Purification, molecular characterization and catalytic properties of a Pseudomonas fluorescens enzyme having cholinesterase-like activity.

An enzyme with a cholinesterase (ChE) activity, produced by Pseudomonas fluorescens, was purified to homogeneity in a three-step procedure. Analysis by non-denaturing and SDS-PAGE, and by isoelectric focusing, indicated that the enzyme was a monomer of 43 kDa, with a pI of 6.1. The N-terminal sequence, AEPLKAVGAGEGQLDIVAWPGYIEA, showed some similarities with proteins of the ChE family and a strong similarity with a protein from Escherichia coli with unknown structure and function. Cholinesterase activity at pH 7.0 and 25 degreesC was maximum with propionylthiocholine as substrate (kcat,app=670 min-1), followed by acetylthiocholine, and significantly lower with butyrylthiocholine. Catalytic specificity (kcat/Km) was the same for propionylthiocholine and acetylthiocholine, but was two orders of magnitude lower for butyrylthiocholine. Kinetics of thiocholine ester hydrolysis showed inhibition by excess substrate which was ascribed to binding of a second substrate molecule, leading to non-productive ternary complex (Km=35 microM, KSS=0.49 mM with propionylthiocholine). There was low or no reactivity with organophosphates and carbamates. The enzyme inhibited by echothiophate (kII=0.44x102 M-1 min-1) was not reactivated by pralidoxime methiodide. However, the P. fluorescens enzyme had affinity for procainamide and decamethonium, two reversible ChE inhibitors used as affinity chromatography ligand and eluant, respectively. Although similarity of the N-terminal amino acid sequence of the enzyme with an internal sequence of ChEs is weak, its catalytic activity towards thiocholine esters, and its affinity for positively charged ligands supports the contention that this enzyme may belong to the ChE family. However, we cannot rule out that the enzyme belongs to another structural family of proteins having cholinesterase-like properties. The reaction of the enzyme with organophosphates suggests that it is a serine esterase, and currently this enzyme may be termed as having a cholinesterase-like activity.

Cholinesterases from the common oyster (Crassostrea gigas). Evidence for the presence of a soluble acetylcholinesterase insensitive to organophosphate and carbamate inhibitors.

Marine bivalves such as oysters and mussels are widely used as bioindicators of contamination in the monitoring of pollutant effects. As filter feeders, these species are known to be good general indicators of chemical contamination. However, the efficient use of decreased acetylcholinesterase activity in the oyster as a biomarker of exposure to neurotoxic compounds requires a definition of the different types of cholinesterases coexisting in this mollusk. This study reports the partial purification, separation and characterization of two cholinesterases extracted from the oyster Crassostrea gigas. Differences in apparent molecular weight, type of glycosylation and hydrophobicity, and sensitivity to inhibitors suggest that they are encoded by two different genes. 'A' cholinesterase (apparent molecular weight 200 kDa) is anchored to the membrane via a glycolipid, is not glycosylated but sensitive to organophosphate and carbamate inhibitors. 'B' cholinesterase (molecular weight 330 kDa) is hydrophilic, glycosylated and highly resistant to organophosphate and carbamate inhibitors. The kinetic properties of these two cholinesterases were compared with those of other invertebrate cholinesterases. The presence of a cholinesterase insensitive to insecticides suggests that a significant improvement in the use of oyster cholinesterases as biomarkers of pollutant effects could be achieved by simple separation of the two forms.

[A comparative study of the cholinesterase and carboxylesterase sensitivities of mammals and arthropods to new phenyl and glycidyl esters of dialkyl thiophosphoric acid]. / Sravnitel'noe issledovanie chuvstvitel'nosti kholinesteraz i karboksilésteraz nekotorykh mlekopitaiushchikh i chlenistonogikh k novym fenilovym i glitsidilovym éfiram dialkiltiofosfornoi kisloty.

The antienzymic activities of 14 organophosphorous compounds, the derivatives of dialkyl thiophosphoric acid, towards the acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and carboxylesterase (CE) from the spring grain aphid and mammals were investigated. The dependence of inhibitory activity of the compounds on their alkyl radical length was shown to be different for the AchE from the aphid and man. Some less pronounced differences in this dependence were revealed between the BuChEs from the aphid and horse as well as between the CEs from the aphid, mouse and red spider mite. The data give evidence of a distinction in structure of the active surfaces of the enzymes from the aphid and mammals. Some peculiar properties of the aphid cholinesterases are discussed taking account of the results of the present and previous papers.

[Cholinesterase].

Since the chromatographic separation of cholinesterase (ChE) by Malström in 1956 many investigator studied ChE isozyme, Harris divided five spots by two dimensional paper electrophoresis and starchgel electrophoresis, and referred as C1 C2 C3 C4. Clinically, Juul separated ChE 12 bands by polyacrylamidegel electrophoresis. We separated ChE as five bands using polyacrylamidegel electrophoresis, revealing fusion and deformity of the band. Takahashi et al reported separation of band using acetyl and butyrylthiocholine as substrate. They found abnormal band in liver cirrhosis, however they have thought it acetyl cholinesterase. Hada et al revealed a defect of band II in liver cirrhosis. They investigated ChE isozyme using affinity electrophoresis with Concanavalin A (Con A) and wheat germ agglutinin (WGA). They found disappearance of band 2, Con A and WGA containing agarose gel electrophoresis seem to be useful method in differentiating liver cirrhosis from chronic hepatitis. The number of isozyme fraction exhibited a species related variations in laboratory animals. Rats, hamsters guinea pigs, rabbits, dogs, monkeys, pigs, horses and quails have 4, 3, 4, 3-5, 3, 3, 4 and 3 isozyme bands, respectively.

A cytochemical study of the nervous system of the proteocephalidean cestode, Proteocephalus pollanicola.

The localization and distribution of cholinergic, serotoninergic and peptidergic nerve elements in the proteocephalidean tapeworm, Proteocephalus pollanicola, have been investigated by enzyme histochemistry, and by an indirect immunofluorescence technique interfaced with confocal scanning laser microscopy. Cholinesterase (ChE) activity was localized in the major components of the central nervous system (CNS) and the peripheral nervous system (PNS), including the innervation of the reproductive structures of the worm. Serotoninergic (5-HT) nerves were found in the paired cerebral ganglia, transverse commissure and in the 10 longitudinal nerve cords. Antisera to 17 mammalian regulatory peptides and the invertebrate peptide FMRFamide have been used to explore the peptidergic nervous system of the worm. The most extensive immunostaining occurred with antisera raised to members of the neuropeptide Y superfamily, namely neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP). In all cases, intense immunoreactivity was found in numerous cell bodies and fibres of both the CNS and PNS, including the innervation of the reproductive apparatus. FMRFamide antisera stained the same structures to a comparable degree as those raised to the NPY superfamily. Cholinergic and peptidergic elements were much more prevalent within the CNS, while the serotoninergic nerve fibres tended to dominate in the PNS. The overlap obtained in staining patterns for the peptidergic and cholinergic components suggests that there may be a certain amount of co-localization of peptides with small-molecule transmitter substances in the same neurone. Weak staining for the tachykinin, substance P and for calcitonin gene-related peptide (CGRP) was confined to the major longitudinal nerve cords.

Dimeric forms of cholinesterase in Sipunculus nudus.

In developing a research on the cholinesterase (ChE) evolution in Invertebrata, this enzyme was studied in the unsegmented marine worm Sipunculus nudus. ChE activity was solubilized through three successive steps of extraction. These fractions are noted as low-salt (LSS), detergent (DS) and high-salt soluble (HSS) and represent 27%, 68% and 5% of total activity, respectively. LSS and DS ChE were purified to homogeneity by affinity chromatography on edrophonium-Sepharose gel. Purification factors of 1700 (LSS) and 1090 (DS) were obtained. The small amount of HSS ChE prevented a similar purification and an extensive characterization. Based on SDS/PAGE and density-gradient centrifugation, both LSS and DS enzymes show a M(r) value of about 130,000 and are likely G2 globular dimers of a 67,000 subunit. Moreover, LSS ChE seems to be an amphiphilic form including a hydrophobic domain, while DS ChE is probably linked to the cell membrane by a phosphatidylinositol anchor. Both LSS and DS enzymes hydrolyze at the highest rate propionylthiocholine. However, they also show a fairly high catalytic efficiency with other thiocholine esters as substrates, thus suggesting a wide and little-specialized conformation of the active site. Based on immunological cross-reactivity trials, LSS and DS ChE from S. nudus show a reduced structural affinity with a molluscan (Murex brandaris) enzyme. HSS ChE, an acetylcholinesterase, is also solubilized by heparin, like typical vertebrate HSS asymmetric enzymes. However, it lacks fast-sedimenting forms and an enzyme-anchoring collagenous structure.

The cholinergic, serotoninergic and peptidergic components of the nervous system of Moniezia expansa (Cestoda, Cyclophyllidea).

The central (CNS) and peripheral (PNS) nervous systems of the cyclophyllidean tapeworm, Moniezia expansa, were examined for the presence of cholinergic, serotoninergic and peptidergic elements using enzyme cytochemical and immunocytochemical techniques in conjunction with light and confocal scanning laser microscopy. Cholinesterase activity and 5-hydroxytryptamine- and regulatory peptide-immunoreactivities (IRs) were localized to the nerve fibres and cell bodies of all of the major neuronal components in the CNS of the worm, including the cerebral ganglia and connecting commissure, the 10 longitudinal nerve cords and associated transverse ring commissures. Although each of the 3 systems appeared well developed and comprised a significant portion of the nervous system, the serotoninergic constituent was the most highly developed, consisting of a vast array of nerve fibres and cell bodies distributed throughout the strobila of the worm. A close association of cholinesterase reactivity and peptide-IRs was evident throughout the CNS, indicating the possible co-localization of acetylcholine and neuropeptides. Within the PNS, cholinergic activity and serotoninergic- and peptidergic-IRs occurred in the subtegumental network of nerve fibres and somatic musculature. Although all 3 neurochemical elements were present in the acetabula, they were found in different nerve fibres; only cholinergic and peptidergic cell bodies were found. The common genital opening, vagina and ootype regions of the reproductive system displayed a rich innervation of all 3 types of neuronal populations. Within the peptidergic system, immunostaining with antisera raised to the C-terminus of the neuropeptide Y superfamily of peptides and the invertebrate peptides, neuropeptide F (M. expansa) and FMRFamide was the most prevalent. Limited positive-IR for substance P and neurokinin A were also recorded in the CNS of the worm.

Protease inhibitors and indoleamines selectively inhibit cholinesterases in the histopathologic structures of Alzheimer disease.

Neurofibrillary tangles and amyloid plaques express acetylcholinesterase and butyrylcholinesterase activity in Alzheimer disease. We previously reported that traditional acetylcholinesterase inhibitors such as BW284C51, tacrine, and physostigmine were more potent inhibitors of the acetylcholinesterase in normal axons and cell bodies than of the acetylcholinesterase in plaques and tangles. We now report that the reverse pattern is seen with indoleamines (such as serotonin and its precursor 5-hydroxytryptophan), carboxypeptidase inhibitor, and the nonspecific protease inhibitor bacitracin. These substances are more potent inhibitors of the cholinesterases in plaques and tangles than of those in normal axons and cell bodies. These results show that the enzymatic properties of plaque and tangle-associated cholinesterases diverge from those of normal axons and cell bodies. The selective susceptibility to bacitracin and carboxypeptidase inhibitor indicates that the catalytic sites of plaque and tangle-bound cholinesterases are more closely associated with peptidase or protease-like properties than the catalytic sites of cholinesterases in normal axons and cell bodies. This shift in enzymatic affinity may lead to the abnormal protein processing that is thought to play a major role in the pathogenesis of Alzheimer disease. The availability of pharmacological and dietary means for altering brain indoleamines raises therapeutic possibilities for inhibiting the abnormal cholinesterase activity associated with Alzheimer disease.

Separation in a single step by affinity chromatography of cholinesterases differing in subunit number.

We describe an affinity chromatography method in which dimethylaminoethylbenzoic acid-Sepharose 4B is used, making it possible to separate in one step the molecular forms of globular acetylcholinesterase (AChE, EC 3.1.1.7) or butyrylcholinesterase (ChE, EC 3.1.1.8). A crude extract containing these enzymes was deposited onto the chromatography gel, washed, and eluted by a linear gradient of tetramethylammonium chloride (0-0.3 M). With rat brain AChE, two well-separated peaks were eluted in the presence of 1% Triton X-100; the first peak corresponded to 4 S forms and the second to 11 S forms. This separation was very efficient for salt-soluble activity and less efficient for the detergent-soluble AChE. In this case, the 4 S peak represented only 6.5% of total detergent-soluble activity and was cross-contaminated by the 11 S form. Rat serum ChE was efficiently separated into two peaks of 7 S and 11 S. This method could potentially be adapted to separate other multimeric proteins with varying numbers of affinity sites.

Phosphonylation of purified human, canine and porcine cholinesterase by soman. Stereoselective aspects.

Cholinesterases (EC 3.1.1.8, acylcholine acylhydrolase) from the sera of man, dog and pig were purified 400-600-fold using a combination of ion-exchange and affinity chromatography. In a first approach, phosphonylation by soman was studied by using the half-resolved epimers C(+)P(+/-)-soman and C(-)P(+/-)-soman. The degradation of soman at the nanomolar level was followed in time by determining the remaining soman by capillary gas chromatography with NP detection. In the three sera investigated the P-(-)-epimer phosphonylates at a higher rate than its corresponding P(+)-counterpart and the stereoselectivity is greater for the C(+)-epimers than for the C(-)-epimers. Individual soman isomers were isolated from C(+)- and C(-)-epimers and quantified by gas chromatography. Second-order rate constants were determined for the phosphonylation of purified cholinesterase by isolated soman isomers. The C(+)P(-)-isomer has the highest phosphonylation rate for the three species; the other toxic isomer, C(-)P(-), has a five to ten-fold lower rate. The overall stereoselectivity is more marked in human cholinesterase than in canine. Porcine serum cholinesterase is phosphonylated by the P(-)-isomers at a slightly higher rate than the human enzyme.

[An enzyme from the fungus Aspergillus niger that hydrolyzes choline ethers]. / Ferment iz griba Aspergillus niger, gidrolizuiushchii kholinovye éfiry.

The acetylthiocholine-hydrolyzing enzymatic activity inhibited by the neostigmine and partly physostigmine has been found in extracts from mycelium of fungus Aspergillus niger. The enzyme has been isolated and 15-20 fold purified. The cholinesterase activity of the protein (Kmu 7.10-7 M) is comparable with known for analogous enzymes from higher plants, for its inhibition high concentrations of substrate (greater than 10-3M) are required. The enzyme hydrolyzes acetylthiocholine with rate approximately 1.5 times higher than butyrylthiocholine. Molecular mass of native protein is approximately 600 kDa, subunits -63 and 44 kDa.

Histo-electrophoretic analysis of the microheterogeneity and region-specificity of isozymes.

An optimal and practical method for high resolution separation and visualization of soluble and membrane-binding isoenzymes is described. By use of this method, non-specific esterase, lactate dehydrogenase and acid phosphatase from different tissues of mice can be separated into more than 50, 30 and 20 components, respectively. Cholinesterase, glucose-6-phosphate dehydrogenase, acid phosphatase, succinate dehydrogenase, lactate dehydrogenase and non-specific esterase isolated from the lumbar spinal cord of chick embryos at different ages can be separated into about 15, 12, 9, 6, 14 and 20 components, respectively. In addition, an attempt has been made to evaluate quantitatively by densitometry differences in enzyme activity among different regions of the same tissue (e.g. spinal cord). The findings revealed that there are differences in isoenzyme components between the dorsal and ventral regions of the spinal cord. Collectively, the combination of direct tissue isoelectric focusing and fast enzyme visualization reveals a greater number of isoenzyme components than can be demonstrated by other means. This method is an extremely useful procedure for understanding and analyzing the nature and topographic localization of isoenzyme components.