Title: Association of tetramers of human butyrylcholinesterase is mediated by conserved aromatic residues of the carboxy terminus Altamirano CV, Lockridge O Ref: Chemico-Biological Interactions, 119-120:53, 1999 : PubMed
Human butyrylcholinesterase (BChE) is composed predominantly of tetramers. Our laboratory has shown that up to 40 carboxy terminal residues of each subunit contribute to the stabilization of tetramers (R.M. Blong, E. Bedows, O. Lockridge, The tetramerization domain of butyrylcholinesterase is at the carboxy-terminus, Biochem. J. 327 (1997) 747-757). To better define the residues which participate in tetramer stabilization, the in vivo interaction of the BChE C-terminus 46 residue peptide was quantitated for wild type and mutant BChE using the yeast two-hybrid system. The wild type C-terminal peptides interacted with one another in this system. The K-variant (A539T) and C571A peptides showed interaction similar to that of the wild type. However, only 11.7% of the interaction seen with the wild type peptide was observed with the mutant in which seven conserved aromatic residues (Trp 543, Phe 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564) had been altered to alanines (aromatics off mutant). When these seven mutations were incorporated into the complete BChE molecule and expressed in 293T cells, only monomers and dimers were observed. The addition of poly-L-proline to the medium of 293T cells expressing wild type BChE resulted in the increase of the tetrameric form, similar to that observed by Bon et al. (S. Bon, F. Coussen, J. Massoulie, Quaternary associations of acetylcholinesterase II. The polyproline attachment domain of the collagen tail, J. Biol. Chem. 272 (1997) 3016-3021) for acetylcholinesterase expressed in COS cells. However, no increase in tetramers was observed with poly-L-proline addition to the medium of 293T cells expressing the aromatics off BChE mutant. These observations suggest that the stabilization of BChE tetramers is mediated through the interaction of the seven conserved aromatic residues, Trp 543, Phe 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564, and that the poly-L-proline induced increase in tetrameric BChE is mediated through these seven aromatic residues.
Soluble extracts of chicken peripheral nerve contain detectable amounts of phenyl valerate esterase (PVase) activity (about 2000 nmol/min per g of fresh tissue). More than 95% of this activity is inhibited in assays where substrate has been added to a preincubated mixture of tissue with the non-neuropathic organophosphorus compound (OP) paraoxon (O,O'-diethyl p-nitrophenyl phosphate): residual activity includes soluble neuropathy target esterase (S-NTE) which, by definition, is considered resistant to long-term progressive (covalent) inhibition by paraoxon. However we have previously shown that paraoxon strongly interacts with S-NTE so interfering with its sensitivity to other inhibitors. We now show that, surprisingly, removal of paraoxon by ultrafiltration ('P' tissue) in order to avoid such an interference results in the reappearance of about 65% of total original soluble PVase activity which is inhibited in the presence of this OP. Although a purely reversible non-progressive inhibition might be suspected, kinetic analysis data show a time-progressive inhibition which suggests that such PVase(s) covalently bind paraoxon. Also a time-dependent recovery due to spontaneous reactivation of the PVase activity was observed after dilution of the inhibitor. Gel filtration chromatography of 'P' tissue in Sephacryl S-300 shows that the reactivated activity is associated with proteins of about 100-kDa mass which include S-NTE and an, as yet, unknown number of other PVases. The implications of these findings in the definition of NTE in a target tissue for the so-called organophosphorus-induced delayed polyneuropathy (OPIDP) are discussed.
A novel mouse liver soluble fraction DFPase which has organophosphatase activities with sarin, soman and tabun, was purified and characterized. However, it lacks paraoxonase and arylesterase activities with paraoxon and phenyl acetate, respectively. This DFPase closely resembles and may be identical with the one purified by Little et al. in 1989 from the soluble fraction of rat liver, based on its substrate specificity, size (approximately 39 kDa) and its stimulation by several metal ions, namely magnesium, manganese and cobalt. Sequencing of our purified mouse liver DFPase showed it to be identical in its amino acid sequence with the recently identified senescence marker protein-30 (SMP-30) by Fujita et al. in 1996. Other senescence marker proteins possessing high structural homology with the mouse SMP-30 have also been found and sequenced from human and rat livers. There is no structural homology between the senescence marker protein family and the group of mammalian paraoxonases. Thus, it is clear that there are at least two distinct, unrelated families of mammalian liver enzymes that share DFPase activity.
        
Title: Protein engineering of a human enzyme that hydrolyzes V and G nerve agents: design, construction and characterization Broomfield CA, Lockridge O, Millard CB Ref: Chemico-Biological Interactions, 119-120:413, 1999 : PubMed
Because of deficiencies in the present treatments for organophosphorus anticholinesterase poisoning, we are attempting to develop a catalytic scavenger that can be administered as prophylactic protection. Currently known enzymes are inadequate for this purpose because they have weak binding and slow turnover, so we are trying to make an appropriate enzyme by protein engineering techniques. One butyrylcholinesterase mutant, G117H, has the desired type of activity but reacts much too slowly. This communication describes an attempt to determine the reason for the slow reaction so that a more efficient enzyme might be designed. The results indicate that the mutation at residue 117 has resulted in a distortion of the transition state of the reaction of organophosphorus compounds with the active site serine. This information will be used to develop other mutants that avoid transition state stabilization sites.
        
Title: Kinetic parameters of cholinesterase interactions with organophosphates: retrieval and comparison tools available through ESTHER database: ESTerases, alpha/beta Hydrolase Enzymes and Relatives Chatonnet A, Hotelier T, Cousin X Ref: Chemico-Biological Interactions, 119-120:567, 1999 : PubMed
Cholinesterases are targets for organophosphorus compounds which are used as insecticides, chemical warfare agents and drugs for the treatment of disease such as glaucoma, or parasitic infections. The widespread use of these chemicals explains the growing of this area of research and the ever increasing number of sequences, structures, or biochemical data available. Future advances will depend upon effective management of existing information as well as upon creation of new knowledge. The ESTHER database goal is to facilitate retrieval and comparison of data about structure and function of proteins presenting the alpha/beta hydrolase fold. Protein engineering and in vitro production of enzymes allow direct comparison of biochemical parameters. Kinetic parameters of enzymatic reactions are now included in the database. These parameters can be searched and compared with a table construction tool. ESTHER can be reached through internet (http://www.ensam.inra.fr/cholinesterase). The full database or the specialised X-window Client-server system can be downloaded from our ftp server (ftp://ftp.toulouse.inra.fr./pub/esther). Forms can be used to send updates or corrections directly from the web.
        
Title: Organophosphate inhibition of human heart muscle cholinesterase isoenzymes Chemnitius JM, Sadowski R, Winkel H, Zech R Ref: Chemico-Biological Interactions, 119-120:183, 1999 : PubMed
The rate of acetylcholine hydrolysis of mammalian heart muscle influences cardiac responses to vagal innervation. We characterized cholinesterases of human left ventricular heart muscle with respect to both substrate specificity and irreversible inhibition kinetics with the organophosphorus inhibitor N,N'-di-isopropylphosphorodiamidic fluoride (mipafox). Specimens were obtained postmortem from three men and four women (61 +/- 5 years) with no history of cardiovascular disease. Myocardial choline ester hydrolyzing activity was determined with acetylthiocholine (ASCh; 1.25 mM), acetyl-beta-methylthiocholine (AbetaMSCh; 2.0 mM), and butyrylthiocholine (BSCh; 30 mM). After irreversible and covalent inhibition (60 min; 25 degrees C) with a wide range of mipafox concentrations (50 nM-5 mM), residual choline ester hydrolyzing activities were fitted to a sum of up to five exponentials using weighted least-squares non-linear curve fitting. In each ease, quality of curve fitting reached its optimum on the basis of a four component model. Final classification of heart muscle cholinesterases was achieved according to substrate hydrolysis patterns (nmol/min per g wet weight) and to second-order organophosphate inhibition rate constants k2 (1/mol per min); one choline ester hydrolyzing enzyme was identified as acetylcholinesterase (AChE; k2/mipafox = 6.1 (+/- 0.8) x 10(2)), and one as butyrylcholinesterase (BChE; k2/mipafox = 5.3 (+/- 1.1) x 10(3)). An enzyme exhibiting both ChE-like substrate specificity and relative resistance to mipafox inhibition (k2/mipafox = 5.2 (+/- 1.0) x 10(-1)) was classified as atypical cholinesterase.
Enzymes catalyzing the hydrolysis of highly toxic organophosphorus compounds (OPs) are classified as organophosphorus acid anhydrolases (OPAA; EC 3.1.8.2). Recently, the genes encoding OPAA from two species of Alteromonas were cloned and sequenced. Sequence and biochemical analyses of the cloned genes and enzymes have established Alteromonas OPAAs to be prolidases (E.C. 3.4.13.9), a type of dipeptidase hydrolyzing dipeptides with a prolyl residue in the carboxyl-terminal position (X-Pro). Alteromonas prolidases hydrolyze a broad range of G-type chemical warfare (CW) nerve agents. Efforts to over-produce a prolidase from A. sp.JD6.5 with the goal of developing strategies for long-term storage and decontamination have been successfully achieved. Large-scale production of this G-agent degrading enzyme is now feasible with the availability of an over-producing recombinant cell line. Use of this enzyme for development of a safe and non-corrosive decontamination system is discussed.
        
Title: The role of paraoxonase (PON1) in the detoxication of organophosphates and its human polymorphism Costa LG, Li WF, Richter RJ, Shih DM, Lusis A, Furlong CE Ref: Chemico-Biological Interactions, 119-120:429, 1999 : PubMed
In human populations, serum paraoxonase (PON1) exhibits a substrate dependent polymorphism. The Arg192 isoform hydrolyzes paraoxon rapidly but diazoxon, soman and especially sarin slowly. On the other hand, the Gln192 isoform hydrolyzes paraoxon slowly, but diazoxon, soman and sarin more rapidly than the Arg192 isoform. Our experiments with a mouse model system have convincingly shown that PON1 plays a major role in the detoxication of organophosphate (OP) compounds processed through the P450/PON1 pathway. Recent studies have also shown that PON1 plays an important role in the metabolism of oxidized lipid compounds. Currently, there is an effort underway to identify genes and polymorphisms that play an important role in 'environmental susceptibility'. The PON1 polymorphism has been cited as a prime example of such a genetic polymorphism. The advent of the polymerase chain reaction (PCR) for DNA amplification with improvements, modifications and automation has provided a very convenient way to do individual genotyping. It is tempting to set up large scale PCR analyses of populations to determine individuals at risk for environmental exposures affected by the PON1 polymorphism. In fact, a number of such studies have already been carried out in examining the relationship of the PON1 polymorphism to vascular disease. We advocate the use of a high throughput two-dimensional enzyme assay that provides both PON1 genotype and phenotype (PON1 status). The high level of variation of gene expression within each genetic class in humans, together with our animal model studies indicate that it is very important to determine PON status as opposed to PON1 genotype alone. Experiments in rats and mice have shown that injection of PON1 purified from rabbit serum by the i.v., i.p. or i.m. route, significantly increases PON1 activities in rodents' plasma. Under these conditions, the acute toxicity (assessed by the degree of acetylcholinesterase inhibition) of paraoxon and chlorpyrifos oxon is significantly decreased, compared to control animals. Protection is maximal when PON1 is administered before the OPs, but still occurs when PON1 is utilized as a post-exposure treatment. Furthermore, protection by PON1 is also provided toward the parent compound chlorpyrifos. Pon1-knockout mice display a much greater sensitivity to chlorpyrifos oxon toxicity than wild mice. However, the acute toxicity of guthion, which is not a substrate for PON1, does not differ between knockout and wild mice. These observations underline the importance of considering both genetic variability of enzyme isoform as well as enzyme level (PON1 status) and the developmental time course of appearance of PON1 in developing risk assessment models.
        
Title: Different role of carboxylesterases in toxicity and tolerance to paraoxon and DFP Dettbarn WD, Yang ZP, Milatovic D Ref: Chemico-Biological Interactions, 119-120:445, 1999 : PubMed
The contribution of carboxylesterase (CarbE) to toxicity and tolerance to the organophosphorus anticholinesterases (OP-antiChE) paraoxon (diethyl p-nitrophenyl phosphate) and DFP (diisopropylphosphorofluoridate) was investigated in rats. Daily injections (20 days) of paraoxon (0.33 micromol/kg) or DFP (2.72 micromol/kg) reduced AChE activity in brain to 29 or 16% and in diaphragm to 58 or 54%, respectively. The animals tolerated an accumulated 6-fold LD50 dose and survived an LD90 dose of carbachol, indicating tolerance to this cholinergic agonist. A single dose of paraoxon or DFP significantly reduced CarbE activity of plasma, lung and liver. After paraoxon, rapid recovery was seen of plasma and liver CarbE while recovery after DFP was much slower. Daily pretreatment with the CarbE inhibitors CBDP (2-[o-cresyl]-4H-1,2,3-benzodioxa- phosphorin-2-oxide) (7.22 micromol/kg, s.c.) or iso-OMPA (tetraisopropylpyrophosphoramide) (8.76 micromol/kg, i.p.), followed by paraoxon (0.33 micromol/kg, s.c.) 30 min later, prevented the development of tolerance to paraoxon and potentiated its toxicity. Rats died on day four of the combined treatment. The CarbE inhibitors neither potentiated the DFP toxicity, nor prevented tolerance development to DFP. We conclude that rat plasma CarbE provides a significant protection against paraoxon toxicity because its rapid reactivation can reduce the toxicity of repeated paraoxon applications and thus contribute to tolerance development. This same mechanism does not apply to DFP toxicity, as inhibition of CarbE of plasma, liver and lung neither potentiated its toxicity, nor prevented tolerance development. These findings confirm previous observations that CarbE detoxification is of greater importance for highly toxic OP-antiChEs such as nerve agents and paraoxon than for less toxic ones such as DFP.
        
Title: Rational design of organophosphorus hydrolase for altered substrate specificities Di Sioudi BD, Miller CE, Lai K, Grimsley JK, Wild JR Ref: Chemico-Biological Interactions, 119-120:211, 1999 : PubMed
Organophosphorus hydrolase (OPH) is a bacterial enzyme that hydrolyzes a broad variety of OP neurotoxins, including chemical warfare agents and many widely used pesticides. OPH has extremely high hydrolytic efficiency with different phosphotriester and phophothiolester pesticides (k(cat) = 50-15,000 s(-1)) as well as phosphorofluorates such as DFP and the chemical warfare agents sarin and soman (k(cat) = 50-11,000 s(-1)). In contrast, the enzyme has much lower catalytic capabilities for phosphonothioate neurotoxins such as acephate or the chemical warfare agent VX [O-ethyl S-(2-diisopropyl aminoethyl) methylphosphonothioate] (k(cat) = 0.3-20 s(-1)). Different metal-associated forms of the enzyme have demonstrated varying hydrolytic capabilities for each of the OP neurotoxins, and the activity of OPH (Co2+) is consistently higher than that of OPH (Zn2+) by five- to 20-fold. Protein engineering strategies have exploited these metal-induced catalytic differences, and other slight modifications to the opd gene have resulted in significant enhancement of the rates of detoxification of the thioate pesticides and chemical warfare agents. In order to develop practical applications of OPH, other experiments have focused on improvement of enzyme production, localization, stability, and shelf-life, as well as efficient catalysis of substrates of interest.
        
Title: Evidence that several conserved histidine residues are required for hydrolytic activity of human paraoxonase/arylesterase Doorn JA, Sorenson RC, Billecke SS, Hsu C, La Du BN Ref: Chemico-Biological Interactions, 119-120:235, 1999 : PubMed
Recent evidence has been acquired that implicates an important role for several histidine residues in the hydrolytic mechanisms of human paraoxonase/arylesterase (PON1). Following titration with diethylpyrocarbonate (DEPC), both human serum and recombinant human type Q PON1 were inhibited in respect to their hydrolytic activity in a dose-responsive manner. Human PON1 treated with varying concentrations lost hydrolytic activity, and with each histidine modified, there was an exponential drop in hydrolytic activity. The reaction was followed spectrophotometrically at 244 nm. Recombinant wild-type and C283A PON1 enzymes inhibited with DEPC and subsequently treated with hydroxylamine had partial restoration of activity. The C283A mutant lacks a free sulfhydryl group, indicating that its inactivation is due to histidine specific modification. The dose response and time course of inactivation as well as the extent of reactivation by hydroxylamine were similar for both the wild-type and mutant recombinant enzymes. Mutants of PON1 containing an asparagine substituted for each of several conserved histidine residues lost hydrolytic activity for each single substitution. The mutants of PON1 constructed and assayed for arylesterase activity were H114N, H133N, and H284N. Each single aminoacid substitution rendered the enzyme catalytically inactive. These two pieces of evidence implicate an important role for several histidine residues in the hydrolytic mechanism of PON1. Although it is unusual for a calcium dependent enzyme to require histidines for its catalytic activity, acquired data suggest such a circumstance.
        
Title: Biological variations of human serum butyrylcholinesterase activity in a population from Zagreb, Croatia Flegar-Mestric Z, Surina B, Siftar Z Ref: Chemico-Biological Interactions, 119-120:193, 1999 : PubMed
Biological variations of total butyrylcholinesterase activity (EC 3.1.1.8) in sera were determined in 993 healthy school children (age 8-19 years) and 2246 healthy adults (20-70 years) in a population from Zagreb, Croatia. Physiological variations corresponding to age and sex and genetically determined changes were studied as the important factors affecting biological variation in total butyrylcholinesterase activity. Based on biological variability, using a non-parametric statistical method, reference intervals for total butyrylcholinesterase activity were produced in order to provide medically reliable evaluation of laboratory results by pediatricians and clinicians in our country.
New findings regarding acetylcholinesterase (AChE) in the neuromuscular junction (NMJ), obtained in the last decade, are briefly reviewed. AChE is highly concentrated in the NMJs of vertebrates. Its location remains stable after denervation in mature rat muscles but not in early postnatal muscles. Agrin in the synaptic basal lamina is able to induce sarcolemmal differentiations accumulating AChE even in the absence of a nerve ending. Asymmetric A12 AChE form is the major molecular form of AChE in vertebrate NMJs. Extrajunctional suppression of this form is a developmental phenomenon. Motor nerve is able to reinduce expression of the A12 AChE form in the ectopic NMJs even in muscles with complete extrajunctional suppression of this form. The 'tail' of the A12 AChE form is made of collagen Q. It contains domains for binding AChE to basal lamina with ionic and covalent interactions. Muscle activity is required for normal AChE expression in muscles and its accumulation in the NMJs. In addition, the pattern of muscle activation also regulates AChE activity in the NMJs, demonstrating that the pattern of synaptic transmission is able to modulate one of the key synaptic components.
        
Title: Molecular cloning of neuropathy target esterase (NTE) Glynn P, Read DJ, Lush MJ, Li Y, Atkins J Ref: Chemico-Biological Interactions, 119-120:513, 1999 : PubMed
Covalent modification of NTE, a neuronal protein with serine esterase activity, by certain organophosphates (OP) initiates degeneration of long axons in the peripheral and central nervous system. Simple inhibition of NTE esterase activity does not initiate neuropathy; the latter requires aging of the OP bound to the catalytic serine residue so that a negatively-charged species is left attached to the active site. This may indicate that a non-esterase function of NTE is important for axonal maintenance. We have recently cloned NTE and shown that it is unrelated to any known serine hydrolases but contains a novel C-terminal domain which is conserved from bacteria to man. Furthermore, the catalytic serine is located within this domain at the centre of a helical hydrophobic segment of the polypeptide's secondary structure. The integrity of NTE would be severely compromised by the presence of a negatively-charged organophosphate moiety at this site. Implications for possible higher-order structures and functions for NTE are discussed.
We previously demonstrated that a combination of cholinesterase (ChE) pre-treatment with an oxime is an effective measure against soman and sarin. We describe here a novel approach for the preparation of covalently linked ChEs which are immobilized to a polyurethane matrix. Such preparation of ChE-sponges enhances the stability and usefulness of the enzymes in non-physiological environments. The ChE-sponges, which can be molded to any form, can effectively be used to remove and decontaminate organophosphates (OPs) from surfaces, biological (skin or wounds) or otherwise (clothing or sensitive medical equipment), or the environment. The ChE-sponges retained their catalytic activity under conditions of temperature, time, and drying where the native soluble enzyme would rapidly denature, and can be reused in conjunction with oximes many times. The ChE-sponge in the presence of oxime repeatedly detoxified OPs such as DFP or MEPQ. These developments in ChE technology have extended the applicability of OP scavengers from in vivo protection, to a variety of external detoxification and decontamination schemes. In addition to treatment of OP-contaminated soldiers, the ChE-sponge could protect medical personnel from secondary contamination while attending chemical casualties, and civilians exposed to pesticides or highly toxic nerve agents such as sarin.
Protein expression can be controled at different levels. Understanding acetylcholinesterase (EC. 3.1.1.7, AChE) expression in the living organisms therefore necessitates: (1) determination and mapping of control levels of AChE metabolism; (2) identification of the regulatory factors acting at these levels; and (3) detailed insight into the mechanisms of action of these factors. Here we summarize the results of our studies on the regulation of AChE expression in the mammalian skeletal muscle. Three experimental models were employed: in vitro innervated human muscle, mechanically denervated adult fast rat muscle, and the glucocorticoid treated fast rat muscle. In situ hybridization of AChE mRNA, combined with AChE histochemistry, revealed that different distribution patterns of AChE, observed during in vitro ontogenesis and synaptogenesis of human skeletal muscle, reflect alterations in the distribution of AChE mRNA (Z. Grubic, R. Komel, W.F. Walker, A.F. Miranda, Myoblast fusion and innervation with rat motor nerve alter the distribution of acetylcholinesterase and its mRNA in human muscle cultures, Neuron 14 (1995) 317-327). To study the mechanisms of AChE mRNA loss in denervated adult rat skeletal muscle, we exposed deproteinated AChE mRNA to various subcellular fractions in vitro. Fractions were isolated from the normal and denervated rat sternomastoideus muscle. We found significantly increased, but non-specific AChE mRNA degradation capacities in the three fractions studied, suggesting that increased susceptibility of muscle mRNA to degradation might be at least partly responsible for the decreased AChE mRNA observed under such conditions (K. Zajc-Kreft, S. Kreft, Z. Grubic, Degradation of AChE mRNA in the normal and denervated rat skeletal muscle, Book of Abstracts, The Sixth International Meeting on Cholinesterases, La Jolla, CA, March 20-24, 1998, p. A3.). In adult fast rat muscle, treated chronically with glucocorticoids, we found the fraction of early synthesized AChE molecular forms to be reduced and AChE mRNA unchanged. This observation is consistent with the explanation that translation and/or early post-translational processes are impaired under such conditions (M. Brank, K. Zajc-Kreft, S. Kreft, R. Komel, Z. Grubic, Biogenesis of acetylcholinesterase is impaired, although its mRNA level remains normal, in the glucocorticoid-treated rat skeletal muscle, Eur. J. Biochem. 251 (1998) 374-381). The AChE mRNA level is therefore important but not the only control level of AChE expression in the mammalian skeletal muscle.
The paraoxonase/arylesterase phenotype was measured in a Spanish population as previous studies have reported that the polymorphic variation in serum paraoxonase activity may affect the metabolism of organophosphates in individuals at risk of chronic intoxication. The prevalence of congenital deficiency in serum cholinesterase was also established in order to ascertain whether individuals with a congenital defect would be at a higher risk against a potential organophosphate exposure. We consider it useful to incorporate these two biomarkers into the health programme of agricultural workers with the purpose of monitoring workers who spray organophosphate pesticides, as they provide reliable indications of early-stage effects related to biochemical alterations that might precede overt clinical pictures.
        
Title: Estimation of inhibitory organophosphates with purified pig liver carboxylesterase Heymann E Ref: Chemico-Biological Interactions, 119-120:577, 1999 : PubMed
Organophosphates that inhibit acetylcholinesterase normally also inhibit pig liver carboxylesterase irreversibly. Since this liver esterase is well characterized and easily accessible in large amounts, we propose the use of this enzyme for the quantitation of low concentrations of such organophosphates. The principle of two estimation methods is described. Both methods involve the addition of an unknown amount of organophosphate to an assay mixture of purified esterase, buffer and a low affinity esterase substrate. In the first of these methods, the inhibitor concentration is calculated from the esterase activities before and after the addition of the inhibitor. In the second method, the amounts of inhibitor or of enzyme are changed in several assays, until equimolar conditions can be detected from the observed reaction kinetics. The theoretical background of these methods is discussed and practical examples for the estimation of paraoxon (order of 0.1 nmoles) are given.
        
Title: Stereochemical preferences for chiral substrates by the bacterial phosphotriesterase Hong SB, Raushel FM Ref: Chemico-Biological Interactions, 119-120:225, 1999 : PubMed
The bacterial phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of organophosphate nerve agents such as paraoxon (diethyl p-nitrophenyl phosphate) with a turnover number of approximately 10(4) s(-1). The active site of the enzyme has been shown to be composed of a binuclear Zn2+ complex with a bridging hydroxide. The utilization of chiral phosphotriesters has demonstrated that the overall hydrolytic reaction occurs with net inversion of stereochemistry at the phosphorus center. The stereochemical constraints of the active site have been probed by the synthesis and characterization of paraoxon analogs. One or both of the two ethoxy substituents of paraoxon have been replaced with various combinations of methyl, isopropyl, or phenyl groups. Racemic mixtures and individual enantiomers were tested as substrates for the phosphotriesterase. In general, the kinetic constants (k(cat) and k(cat)/Km) for the (-)-enantiomers were one to two orders of magnitude greater than the (+)-enantiomer. Conversely, acetylcholinesterase was more rapidly inactivated by the (+)-enantiomers than the (-)-enantiomers. These results were examined in the context of the three-dimensional structure of the bacterial phosphotriesterase.
        
Title: Organophosphorus acid anhydrolase in slime mold, duckweed and mung bean: a continuing search for a physiological role and a natural substrate Hoskin FC, Walker JE, Mello CM Ref: Chemico-Biological Interactions, 119-120:399, 1999 : PubMed
Recently, and for the first time, a diisopropylphosphorofluoridate (DFP)-hydrolyzing enzyme, i.e. an organophosphorus acid anhydrolase (OPAA), has been reported in a plant-source. Based on this and other suggestive evidence, the ability of three plant sources and a protist to hydrolyze DFP and 1,2,2-trimethylpropyl methylphosphonofluoridate (Soman) were tested, and the effects of Mn2+ and ethylenediamine tetraacetate (EDTA) on this activity. The plants are duckweed (Lemna minor), giant duckweed (Spirodela oligorhiza), and germinated mung bean (Vigna radiata); the protist is a slime mold (Dictyostelium discoidium). The tests are based on a crude classification of OPAAs as 'squid type' (DFP hydrolyzed more rapidly than Soman) and all of the others termed by us, with questionable justification, as 'Mazur type' (Soman hydrolyzed more rapidly than DFP). Of the two duckweeds, Spirodela oligorhiza hydrolyzes Soman but not DFP, and Lemna minor does not hydrolyze either substrate. In contrast to the report of Yu and Sakurai, mung bean does not hydrolyze DFP and hydrolyzes Soman with a 5-fold stimulation by Mn2+ and a marked inhibition by EDTA. The slime mold hydrolyzes Soman more rapidly than DFP (but does hydrolyze DFP) and the hydrolysis is Mn2+ stimulated. The failure of these plant sources to hydrolyze DFP is similar to the behavior of OPAA from Bacillus stearothermophilus.
        
Title: Degradation of nerve gases by CLECS and cells: kinetics of heterogenous systems Hoskin FC, Walker JE, Stote R Ref: Chemico-Biological Interactions, 119-120:439, 1999 : PubMed
We have reported the enzymatic hydrolysis of phosphoro- and phosphonofluoridates and phosphoro- and phosphonothiolates and -thionates by an organophosphorus hydrolase (OPH) from Pseudomonas diminuta. In screening for other microbial sources of nerve gas hydrolyzing enzymes, it would be convenient, indeed essential, to be able to determine such hydrolyses on intact cells. As a preliminary step to such screening we have measured the hydrolysis of O,O-diisopropyl S-(2-diisopropylaminoethyl) phosphorothiolate (Tetriso) and O,O-diethyl S-(2-ethylthioethyl) phosphorothiolate (Demeton-S; formerly Isosystox) by intact cells and sonicates. The purified OPH has also been cross-linked to itself (CLEC = cross-linked enzyme crystals) and this has also been tested for its ability to hydrolyze Tetriso and Demeton-S. The testing of such heterogenous systems by a spectrophotometric assay (Ellman) has required novel modifications. Our findings are that both Tetriso and Demeton-S are subject to intact-cell assay, that both are readily hydrolyzed by the CLEC-ed OPH without marked change in kinetics, but that at any given substrate concentration Tetriso is hydrolyzed much more rapidly. However, since Demeton-S is commercially available, this appears to be the substrate most suitable for screening for our final goal in a search for sources of enzymes to detoxify O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX).
        
Title: Human serum paraoxonase (PON1): identification of essential amino acid residues by group-selective labelling and site-directed mutagenesis Josse D, Xie W, Masson P, Lockridge O Ref: Chemico-Biological Interactions, 119-120:71, 1999 : PubMed
Human serum paraoxonase/arylesterase (PON1, EC 3.1.8.1.) is a calcium-dependent enzyme which hydrolyzes a wide variety of organophosphates, including paraoxon, DFP, sarin and soman. Although the 3-D structure of PON has not yet been determined and its sequence shows no similarity with any other crystallized proteins, we undertook to identify some of its essential amino acid residues by two complementary approaches: group-specific labelling and site-directed mutagenesis. Group-specific labelling studies, performed on the purified native enzyme, indicated that one or more Trp, His and Asp/Glu are potentially important residues for PON activity. Based on these results, we identified some of these residues, conserved in the sequenced mammalian PON1, by site-directed mutagenesis. PON1 mutants were transiently expressed in 293T cells. The catalytic constants k(cat) and Km (relative to k(cat) and Km of the wild-type) determined with four different substrates (phenylacetate, paraoxon, diazoxon, chlorpyrifos oxon), were not significantly changed for the following mutants: W193A, W201A, W253A, H160N, H245N, H250N, H347N, E32A, E48A, D88A, D107A, D121A, D273A. By contrast, k(cat) was less than 1% for eight mutants: W280A, H114N, H133N, H154N, H242N, H284N, E52A and D53A. The essential amino acid residues identified in this work could be part of the PON1 active site, acting either as calcium ligands (E52 and D53?) or as substrate binding (W280?) or nucleophilic (His residues?) sites. However, we cannot rule out that the effects of mutations on catalytic properties resulted from a remote conformational change and/or misfolding of mutant proteins.
        
Title: Tryptophan residue(s) as major components of the human serum paraoxonase active site Josse D, Xie W, Masson P, Schopfer LM, Lockridge O Ref: Chemico-Biological Interactions, 119-120:79, 1999 : PubMed
Serum paraoxonase (PON1, EC 3.1.8.1.) is a high density lipid- (HDL)-associated, calcium-dependent enzyme whose 3D structure, active site residues and physiological substrates are not known. The kinetic parameters k(cat) and Km (relative to k(cat) and Km of the wild-type), determined with four substrates (phenylacetate, paraoxon, diazoxon and chlorpyrifosoxon) were less than 1, and more than 100% for the W280A and W280F mutant enzymes, respectively. These results indicated that the aromatic/hydrophobic character of the amino acid in position 280 is essential for PON1 activity. In this study, we investigated whether this aromatic residue is in the PON1 active site. Group-specific labelling studies with N-bromosuccinimide, an oxidative agent of tryptophan, strongly suggested that one or several Trp could be in the active site of PON1 but we could not conclude either on the specificity of the labelling reaction or on the number of oxidized Trp. However, although PON activity was not altered by the hydrophilic tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl chloride (NBC), it was significantly reduced by the p-nitrophenylacetate analog 2-acetoxy-5-nitrobenzyl chloride (ANBC), whose hydrolysis by PON1 generated NBC in the active site. Moreover, since at least one calcium ion is present in the PON catalytic site, we attempted to probe the metal local environment using the calcium analog terbium. The luminescence spectrum of the PON terbium complex exhibited an emission peak at 545 nm characteristic of an aromatic residue (Trp and/or Tyr)-terbium interaction. In conclusion, both the results obtained with the mechanism-based inhibitor of PON1 (ANBC) and the calcium-binding site luminescent probe terbium support the hypothesis of the presence of at least one Trp residue in the PON1 active site. Trp residue(s) may be involved in the binding of aromatic substrates.
Cholinesterase inhibitors (anti-ChEs) include a wide range of therapeutic, agricultural and warfare agents all aimed to inhibit the catalytic activity of the acetylcholine (ACh) hydrolysing enzyme acetylcholinesterase (AChE). In addition to promoting immediate excitation of cholinergic neurotransmission through transient elevation of synaptic ACh levels, anti-ChEs exposure is associated with long-term effects reminiscent of post-traumatic stress disorder. This suggested that exposure to anti-ChEs leads to persistent changes in brain proteins and called for exploring the mechanism(s) through which such changes could occur. For this purpose, we established an in vitro system of perfused, sagittal mouse brain slices which sustains authentic transcriptional responses for over 10 h and enables the study of gene regulation under controlled exposure to anti-ChEs. Slices were exposed to either organophosphate or cabamate anti-ChEs, both of which induced within 10 min excessive overexpression of the mRNA encoding the immediate early response transcription factor c-Fos. Twenty minutes later we noted 8-fold increases over control levels in AChE mRNA, accompanied by a 3-fold decrease in the mRNAs encoding for the ACh synthesizing enzyme choline acetyltransferase (ChAT) and the vesicular ACh transporter (VAChT). No changes were detected in synaptophysin mRNA levels. These modulations in gene expression paralleled those taking place under in vivo exposure. Of particular concern is the possibility that feedback processes leading to elevated levels of brain AChE may be similarly associated with low-level exposure to common organophosphorous anti-cholinesterases, and lead to long-term deleterious changes in cognitive functions.
        
Title: Isoesterases related to cell differentiation in plant tissue culture Krsnik Rasol M, Cipcic H, Hagege D Ref: Chemico-Biological Interactions, 119-120:587, 1999 : PubMed
Normal, habituated and transformed in vitro tissue lines of sugar beet (Beta vulgaris L.), horseradish (Armoracia lapathifolia Gilib.) and potato (Solanum tuberosum L.) were studied with regard to isoesterase patterns. Isoenzymes were separated in gradient gels (5-12%) of polyacrylamide and by isoelectric focussing in pH range 4-9. 1- and 2-naphtylacetate were used as substrates of broad spectrum which cover also esterases (arylesterases and carboxylesterases) reacting with organophosphorous compounds. Distinct isoesterase patterns were noticed in sugar beet normal, habituated and crown gall tumour tissues. Horseradish tumour and teratoma, on the contrary, differed only in one anodic isoenzyme. Even the malformed shoots and unorganised tissue of teratoma had the same patterns. In potato tuber tissue, change in isoesterase pattern, characterised by disappearance of a dominant dark area, was observed during tumour development. The gradient gels gave more stable and reproducible isoenzyme patterns than isoelectric focussing.
The hydrolysis of the second messenger cyclic AMP (cAMP) by phosphodiesterase 3 (PDE3) is known to play an important regulatory role in the context of relaxation of cavernous smooth muscle of the penis. Thus, we investigated the PDE3A isoform from penile cavernous tissues of male patients with and without symptoms of erectile dysfunction at the molecular biological level. As revealed by reverse transcriptase polymerase chain reaction, of all tissues of the urogenital tract analyzed the expression of the PDE3A gene was highest in the corpus cavernosum. However, significant differences in the levels of gene expression were not found between the two subgroups of patients. Also, the determined nucleotide sequences of the cloned penile PDE3A cDNAs of all patients were absolutely identical. Surprisingly, some deviations could be detected in the cDNA sequences of PDE3A from human myocard and platelets. The data obtained indicate that neither the expression levels nor the sequence deviations of PDE3A are the main reasons for erectile dysfunction in men.
In recent years several lines of evidence have indicated that serum paraoxonase (PON1), and perhaps other mammalian paraoxonases, act as important guardians against cellular damage from toxic agents, such as organophosphates, oxidized lipids in the plasma low density lipoproteins (LDL), and against bacterial endotoxins. For some of these protective activities but not all, PON1 requires calcium ion. The catalyzed chemical reactions generally seem to be hydrolytic, but for some types of protection this may not be so. Several other metals have very high affinity for PON1 and may displace calcium. Replacement or substitution of calcium by other metals could extend the range of catalytic properties and the substrate specificity of the paraoxonases, as it does for the mammalian DFPases. Although this Third International Meeting on Esterases Reacting with Organophosphorus Compounds focuses on the organophosphatase activities of paraoxonase and related enzymes, it is important to also briefly review some of the current directions in several laboratories searching for additional functions of the paraoxonases to extend our understanding of the properties of this family of enzymes which now seem to have both physiological and toxicological importance.
        
Title: Recommended nomenclature system for the paraoxonases La Du BN, Furlong CE, Reiner E Ref: Chemico-Biological Interactions, 119-120:599, 1999 : PubMed
Title: Promotion of organophosphate induced delayed polyneuropathy by certain esterase inhibitors Lotti M, Moretto A Ref: Chemico-Biological Interactions, 119-120:519, 1999 : PubMed
Certain esterase inhibitors elicit or intensify the clinical expression of various insults to axons. This phenomenon was called promotion of axonopathies because these chemicals are not additive neurotoxicants nor do they interfere with the pharmacokinetics. Characterization of promotion was carried out by using organophosphate induced delayed polyneuropathy (OPIDP) as a model. The search for a physiological explanation of promotion has the following background: (1) Promotion expresses clinically the biochemical lesions which are otherwise well compensated (such as 30/40% neuropathy target esterase (NTE) inhibition by neuropathic organophosphates). (2) Promotion is not specific because axonopathies of different origin are affected. (3) Promoters are effective when given several days before the neuropathic insult. (4) Promotion is less effective in young animals as compared with adults. (5) Promotion occurs when axons, but not necessarily the cell body, are targeted by promoters. (6) Repeated dosing with a promoter failed to produce axonopathy. Based on this evidence it is suggested that promotion might interfere with a mechanism(s) of compensation and/or repair of long axons. The target of promotion of axonopathies is thought to be similar or linked to NTE which is defined as the phenyl valerate esterase activity (PVE) in nervous tissues resistant to paraoxon and sensitive to mipafox (40 and 50 microM, pH 8.0, 20 min, respectively). Mipafox (50 microM) resistant PVEs include some activity sensitive to the promoter phenylmethane sulfonylfluoride (PMSF) but no correlation was found between its inhibition and promotion. A complete titration curve of paraoxon-resistant PVEs by mipafox (0-1 mM) dissected, besides NTE (I50 about 10 microM), another PVE with an I50 of approximately 200 microM. This enzyme was present in hen brain, spinal cord and peripheral nerve, corresponding to about 10, 20 and 30% of NTE activity, respectively, and was sensitive both in vitro and in vivo to promoters and much less so to neuropathic NTE inhibitors. By means of chromatography, other workers have identified in soluble extracts of peripheral nerves two forms of mipafox-sensitive PVEs with different molecular weights and different sensitivity to mipafox. These might correspond to NTE and to the other enzyme. Inhibition in vivo of the latter also correlated with promotion.
        
Title: Role of edrophonium in prevention of the re-inhibition of acetylcholinesterase by phosphorylated oxime Luo C, Saxena A, Ashani Y, Leader H, Radic Z, Taylor P, Doctor BP Ref: Chemico-Biological Interactions, 119-120:129, 1999 : PubMed
We examined the role of edrophonium in the acceleration phenomenon using mouse wild-type and mutant D74N AChE inhibited with 7-(O,O-diethyl-phosphinyloxy)-1-methylquinolinium methylsulfate (DEPQ). With DEPQ-inhibited wild-type mouse acetylcholinesterase (AChE), the reactivation kinetic profile demonstrated one-phase exponential association only when 2-[hydroxyimino methyl]-1-methylpyridinium chloride (2-PAM) and 1-(2-hydroxy-iminomethyl-1-pyridinium)-1-(4-carboxy-aminopyridi nium)-dimethyl ether hydrochloride (HI-6) were used as reactivators. When 1,1[oxybis-methylene)bis[4-(hydroxyimino)methyl] pyridinium dichloride (LuH6) and 1,1-trimethylene bis(4-hydroxyimino methyl) pyridinium dichloride (TMB4) were used, the reactivation kinetic profile was biphasic in nature. Edrophonium had no effect on reactivation by 2-PAM and HI-6, but significantly accelerated LuH6- and TMB4-induced reactivation of DEPQ-inhibited wild-type mouse AChE. Comparison of the initial and overall reactivation rate constants with five oximes indicated that acceleration by edrophonium may be due to the prevention of re-inhibition of the reactivated enzyme by the phosphorylated oxime (POX) produced during the reactivation. With LuH6 and TMB4, about 2.5-fold increase in the reactivation rate constants was observed in the presence of edrophonium, but little or no effect was observed with the other three oximes. The initial reactivation rate constants were 5.4- and 4.2-fold of the overall rate constants with LuH6 and TMB4 as reactivators respectively, however, very little change was found between the initial and overall rate constants with the other three oximes. In experiments with D74N AChE, for which the inhibition potency of charged organophosphate (OP) was two to three orders less than wild-type enzyme, edrophonium had no effect on the reactivation by LuH6 and TMB4 and the time courses of reactivation were monophasic. The data from mutant enzyme substantiate the involvement of edrophonium in protecting POX re-inhibition of reactivated enzyme formed during the reactivation of OP-inhibited AChE.
        
Title: Low serum paraoxonase: a risk factor for atherosclerotic disease? Mackness MI, Durrington PN, Ayub A, Mackness B Ref: Chemico-Biological Interactions, 119-120:389, 1999 : PubMed
Serum paraoxonase (PON1) hydrolyses organophosphate (OP) insecticides and nerve gases and is responsible for determining the selective toxicity of these compounds in mammals. PON1 has two genetic polymorphisms giving rise to amino acid substitutions at position 55 and 192. The 192 polymorphism is the major determinant of the PON1 activity polymorphism towards organophosphates. However, the 55 polymorphism also modulates activity. PON1 also may be a determinant of resistance to the development of atherosclerosis by protecting lipoproteins against oxidative modification perhaps by hydrolysing phospholipid-hydroperoxides. The PON1 polymorphisms are important in determining the capacity of high-density lipoprotein (HDL) to protect low-density lipoprotein (LDL) against oxidative modification in vitro and this may explain the relationship between the PON1 alleles and coronary heart disease in case-control studies.
        
Title: A stable preparation of hen brain neuropathy target esterase for rapid biochemical assessment of neurotoxic potential of organophosphates Makhaeva GF, Malygin VV Ref: Chemico-Biological Interactions, 119-120:551, 1999 : PubMed
Neuropathy target esterase (NTE) is a molecular target for organophosphate-induced delayed neurotoxicity (OPIDN). This enzyme has proved to be an excellent tool for the assessment of neuropathic potential of organophosphates (OP), in particular by comparison of an OP inhibitory activity in vitro against NTE and acetylcholinesterase. A large-scale OP screening for delayed neurotoxicity was largely prevented by the lack of an available stable preparation of NTE. To obtain a stable NTE preparation the influence of intensive freezing and subsequent lyophilization of paraoxon-preinhibited (P2 + P3) hen brain membrane fraction on NTE properties has been studied using two neuropathic OP: mipafox and O,O-dipropyldichlorovinyl phosphate (PrDChVP). It was shown that lyophilization preserved a high NTE specific activity and did not alter the inhibitor characteristics of the enzyme. A long-term storage study showed that lyophilized NTE preparation exhibited inhibitory features actually identical to those of the native enzyme during 1 year and retained rather high specific activity; in this case some loss of NTE specific activity has been observed. Comparative studies of inhibition of the native and lyophilized NTE preparations by a model series of phenyl phosphonates RO(C6H5)P(O)ON=CClCH3 (R = alkyl), demonstrated a good correlation between the values pI50 obtained with both enzyme preparations as well as identical structure-activity relationships for the lyophilized and native enzymes. The results allow the conclusion that the obtained NTE preparation can be used as a standard, stable and readily available source of NTE for assessing the anti-NTE activity of OP.
The role of electrostatics in the function of acetylcholinesterase (AChE) has been investigated by both theoretical and experimental approaches. Second-order rate constants (kE = k(cat)/Km) for acetylthiocholine (ATCh) turnover have been measured as a function of ionic strength of the reaction medium for wild-type and mutant AChEs. Also, binding and dissociation rate constants have been measured as a function of ionic strength for the respective charged and neutral transition state analog inhibitors m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA) and m-(t-butyl)trifluoroacetophenone (TBTFA). Linear free-energy correlations between catalytic rate constants and inhibition constants indicate that kE for ATCh turnover is rate limited by terminal binding events. Comparison of binding rate constants for TMTFA and TBTFA attests to the sizable electrostatic discrimination of AChE. Free energy profiles for cationic ligand release from the active sites of wild-type and mutant AChEs have been calculated via a model that utilizes the structure of T. californica AChE, a spherical ligand, and energy terms that account for electrostatic and van der Waals interactions and chemical potential. These calculations indicate that EA and EI complexes are not bound with respect to electrostatic interactions, which obviates the need for a 'back door' for cationic ligand release. Moreover, the computed energy barriers for ligand release give linear free-energy correlations with log(kE) for substrate turnover, which supports the general correctness of the computational model.
        
Title: Structural and hydration changes in the active site gorge of phosporhylated butyrylcholinesterase accompanying the aging process Masson P, Fortier PL, Albaret C, Clery C, Guerra P, Lockridge O Ref: Chemico-Biological Interactions, 119-120:17, 1999 : PubMed
Wild-type (wt) butyrylcholinesterase (BuChE) and the E197D and D70G mutants were inhibited by diisopropylfluorophosphate (DFP) or soman under standard conditions of pH, temperature and pressure. The effect of hydrostatic and osmotic pressures on the aging process of DFP-phosphorylated enzymes (diisopropylphosphoryl-BuChE (DIP-BuChE)) was investigated. Hydrostatic pressure strongly increased the rate of aging of wt enzyme. The activation volumes (deltaV*) for the dealkylation reaction was -150 ml/mol for DIP-wtBuChE. On the other hand, pressure had little effect on the aging of the DIP-E197D mutant and no effect on the DIP-D70G mutant, indicating that the transition state of the aging reaction (dealkylation of an isoproxy chain) was associated with an extended conformation/hydration change in wtBuChE but not in mutants. The rate of aging decreased with osmotic pressure, supporting the idea that water is important for stabilizing the transition state. Molecular dynamics simulations were performed on the wtDIP adduct to relate the kinetic data to hydration changes in the enzyme active site gorge. The pH dependence of the melting temperature (Tm) of native and soman-wtBuChE, as determined by differential scanning calorimetry (DSC), indicated that the stabilization energy of aged BuChE is mainly due to the salt bridge between protonated H438 and PO-, with pK(H438) = 8.3. Electrophoresis under high pressure up to 2.5 kbar showed that aged wtBuChE did not undergo pressure-induced molten globule transition unlike the native enzyme. This transition was not seen for the mutant enzymes, indicating that mutants are resistant to the penetration of water into their structure. Our results support the conclusion that D70 and E197 are major residues for the water/H-bond network dynamics in the active site gorge of BuChE, both residues acting like valves. In mutant enzymes, mutated residues function like check valves: forced penetration of water in the gorge is difficult, release of water is facilitated.
        
Title: The polymorphism of acetylcholinesterase: post-translational processing, quaternary associations and localization Massoulie J, Anselmet A, Bon S, Krejci E, Legay C, Morel N, Simon S Ref: Chemico-Biological Interactions, 119-120:29, 1999 : PubMed
The molecular forms of acetylcholinesterase (AChE) correspond to various quaternary structures and modes of anchoring of the enzyme. In vertebrates, these molecules are generated from a single gene: the catalytic domain may be associated with several types of C-terminal peptides, that define distinct types of catalytic subunits (AChE(S), AChE(H), AChE(T)) and determine their post-translational maturation. AChE(S) generates soluble monomers, in the venom of Elapid snakes. AChE(H) generates GPI-anchored dimers, in Torpedo muscles and on mammalian blood cells. AChE(T) is the only type of catalytic subunit that exists in all vertebrate cholinesterases; it produces the major forms in adult brain and muscle. AChE(T) generates multiple structures, ranging from monomers and dimers to collagen-tailed and hydrophobic-tailed forms, in which catalytic tetramers are associated with anchoring proteins that attach them to the basal lamina or to cell membranes. In the collagen-tailed forms, AChE(T) subunits are associated with a specific collagen, ColQ, which is encoded by a single gene in mammals. ColQ contains a short peptidic motif, the proline-rich attachment domain (PRAD), that triggers the formation of AChE(T) tetramers, from monomers and dimers. The critical feature of this motif is the presence of a string of prolines, and in fact synthetic polyproline shows a similar capacity to organize AChE(T) tetramers. Although the COLQ gene produces multiple transcripts, it does not generate the hydrophobic tail. P, which anchors AChE in mammalian brain membranes. The coordinated expression of AChE(T) subunits and anchoring proteins determines the pattern of molecular forms and therefore the localization and functionality of the enzyme.
        
Title: Improvements in scavenger protection against organophosphorus agents by modification of cholinesterases Maxwell DM, Saxena A, Gordon RK, Doctor BP Ref: Chemico-Biological Interactions, 119-120:419, 1999 : PubMed
The ability of stoichiometric scavengers, such as ChEs, to protect against a variety of OP agents has been demonstrated in several in vivo models. To improve the detoxification of OP agents by ChEs, several approaches have been recently used to increase the stoichiometry, stability, and in vivo effectiveness of ChEs as OP scavengers. For example, the in vitro stoichiometric neutralization of sarin by AChE was increased from 1:1 to 3200:1 by the addition of the oxime HI-6, while the in vivo stoichiometry was increased to 57:1 in mice by HI-6. The aging rate of soman-inhibited mouse AChE was reduced 12-fold in a mutant AChE (E202Q) which resulted in a two-fold increase in oxime-assisted detoxification of soman. To improve the duration of scavenger protection provided by ChEs, the mean residence times of five tissue-derived and two recombinant ChEs injected i.v. in mice were compared with their oligosaccharide profiles. The mean residence times of these ChEs were found to increase with molecular weight and with the levels of oligosaccharide sialylation. The stability of AChE in non-physiological environments was improved by immobilizing it in a polyurethane foam matrix that allowed AChE to retain enzymatic activity at high temperature (75 degrees C) where soluble enzyme denatured. These developments in scavenger technology have improved the in vivo protection provided by OP scavengers and extended their applicability to provide external decontamination of chemical agents and pesticides.
        
Title: Morphometric characteristics of myonuclear distribution in the normal and denervated fast rat muscle fiber Milanic T, Kunstelj A, Mars T, Grubic Z Ref: Chemico-Biological Interactions, 119-120:321, 1999 : PubMed
Unlike the majority of mammalian tissues, which have mononuclear cells as a basic unit of the tissue composition, skeletal muscle fiber is a polynuclear syncytium. Polynuclear organization offers an additional possibility for the regulation of protein expression: it can also be controlled at the level of myonuclear distribution and specialization. Distribution of myonuclei can be considered as the distribution of genes. Variability in gene concentration may have important impact on the regional differentiation of the muscle fiber, since it leads to different regional concentrations of various gene products including factors controlling their expression. The aim of the present study was: 1) to provide morphometrical data on the myonuclear distribution in the junctional and extrajunctional regions of the normal fast rat muscle fiber, and 2) to analyze, whether morphometrical parameters of nuclear distribution change after mechanical denervation of this muscle. Single muscle fibers were isolated from the normal and denervated fast rat m. sternomastoideus. Their neuromuscular junctions were stained by thiocholine histochemical procedure and myonuclei were fluorescently labeled by Hoechst 33342. Myonuclear distribution in each individual muscle fiber was morphometrically analyzed on the image analyzer. Synaptic concentrations of myonuclei were found to exceed extrasynaptic concentrations by a factor of 17. The number of myonuclei accumulated at the endplates did not change after one or two weeks of denervation, neither did the morphometric parameters of these nuclei. A higher concentration of myonuclei due to muscle atrophy was observed in the extrasynaptic region and the longitudinal axis of these nuclei also became significantly shorter. Unchanged morphometric parameters of the junctional myonuclei after denervation are indicative of either irreversibility of the nerve-induced formation of nuclear clusters in this region or persistence of the factors responsible for their formation and maintenance.
        
Title: Localization of cells expressing AChE mRNA in rat striatum using nonradioactive in situ hybridization Mis K, Zajc-Kreft K, Grubic Z Ref: Chemico-Biological Interactions, 119-120:327, 1999 : PubMed
A better understanding of the role of AChE in mammalian brain requires knowledge of the distribution of AChE synthesizing cells in this tissue. The aim of the present study is to test a nonradioactive approach for the localization of AChE mRNA positive cells in rat striatum. Nonradioactive in situ hybridization has not been used before for the localization of this mRNA in mammalian brain. In order to find optimal conditions for localization, we employed both RNA and oligonucleotide probes. We also examined various prehybridization protocols and approaches. The total number of cells in brain sections was determined by subsequent fluorescent staining of the nuclei. Optimal AChE mRNA localization was obtained with a digoxigenine-labeled RNA probe. We were not able to localize AChE mRNA with nonradioactively 3' end-labeled oligonucleotides. An acetylation step prior to hybridization was found to be essential for optimal signal/background ratios; high nonspecific staining was observed, if this step was omitted. In accordance with reports of other authors, who used radioactive in situ hybridization, we found very low percentages of AChE mRNA-positive cells in striatum, although this area exhibits very high AChE staining. In comparison to radioactive techniques, the nonradioactive approach avoids the risks of radioactivity, and is much less time consuming. In our experiments AChE mRNA localization in striatum was practically the same as that demonstrated previously by radioactive approaches.
The present study shows the existence of both Ca2+-dependent and EDTA-resistant hydrolysing activities against HDCP and paraoxon in the particulate and soluble fractions of hen, rat and rabbit liver. HDCP was more extensively hydrolysed than paraoxon in both subcellular fractions and each of three individuals of the three animal species under study in spite of wide interindividual variations. However the ratio of HDCP versus paraoxon hydrolysing activity (HDCPase/paraoxonase), although within the same order of magnitude, cannot be considered as constant as it ranges one- to seven-fold between individuals of the same species. Also there is no constant ratio of Ca2+-dependent/EDTA-resistant activities. Rabbit liver showed the highest rates of Ca2+-dependent hydrolysis for both organophosphorus compounds whereas the hen paraoxonase activity was not inhibited by EDTA. The stereospecific hydrolysis of HDCP was mostly a Ca2+-dependent one, the S-HDCP isomer being hydrolysed faster than the R-HDCP one. The suggestion is made that HDCP could be conveniently used to measure PTE activity in the liver.
The in vivo effects of the organophosphorus compound (OPC) paraoxon (POX) on blood coagulation of mini pigs were assessed by measuring the partial thromboplastin time (PTT), prothrombin time (PT), fibrinogen, factor V, factor VII, factor VIII, antithrombin III, protein C, and platelet count. The mini pigs were randomly assigned to a POX-treatment group (n = 9) receiving 54 mg POX kg(-1) BW(-1) or the control group (n = 9). Measurements were carried out over a period of 150 min after poisoning. The exposure to POX did not have any influence on measurements of PT, factor VIII, factor VII, factor V, antithrombin III, protein C, or fibrinogen compared to the control group evaluated by rank order test (ROT) during the time of observation (150 min). Changes seen in the intrinsic coagulation followed a biphasic pattern corresponding to an early sympathomimetic phase with PTT-shortening and a decrease of the platelet count, and a late vagal phase, with PTT-prolongation. The hypercoagulability seen in the sympathomimetic phase is probably due to a massive release of catecholamines from the adrenals. Previous studies showed in vitro no coagulation activating effect of POX. The hypocoagulability in the vagal phase shown by the PTT-protongation is probably due to POX influencing platelet function or its inhibition of clotting factors, which are serine proteases, or a combination of the two.
        
Title: Phospholipase A2 (PLA2) activity in mini pigs after acute high dose i.v.-paraoxon (POX) intoxication Petroianu GA, Helfrich U, Globig S, Fisher J, Rufer R Ref: Chemico-Biological Interactions, 119-120:497, 1999 : PubMed
The purpose of the present study was to establish in the mini pig model the effects of paraoxon (POX) on PLA2 activity. Six anesthetized and mechanically ventilated mini pigs were infused over 50 min with 0.3, 1, 3, 9, 27 and 81 mg POX kg(-1) BW(-1) dissolved in ethanol, respectively. The control animal received no POX but the ethanol amount corresponding to the highest POX dose. PLA2 activity measurements were carried out immediately after POX application. Data were analysed with the Mann Whitney-Wilcoxon rank order test. Statistical significance was assumed for P < or = 0.05. Exposure to POX inhibited PLA2 activity to 50.5 +/- 8.9% of baseline activity. The changes seen were not dose-dependent. The dose dependency previously demonstrated in vitro was not reproducible in vivo. This is most probably due to the massive endogenous catecholamine release leading to PLA2 activation. An additional masking effect is due to the (co)administration of drugs needed for anesthesia and cardiovascular support, especially Mg2+. These substances also influence the PLA2 activity.
        
Title: The influence of peripheral site ligands on the reaction of symmetric and chiral organophosphates with wildtype and mutant acetylcholinesterases Radic Z, Taylor P Ref: Chemico-Biological Interactions, 119-120:111, 1999 : PubMed
The rates of inhibition of mouse acetylcholinesterase (AChE) (EC 3.1.1.7) by paraoxon, haloxon, DDVP, and enantiomers of neutral alkyl methylphosphonyl thioates and cationic alkyl methylphosphonyl thiocholines were measured in the presence and absence of AChE peripheral site inhibitors: gallamine, D-tubocurarine, propidium, atropine and derivatives of coumarin. All ligands, except the coumarins, at submillimolar concentrations enhanced the rates of inhibition by neutral organophosphorus compounds (OPs) while inhibition rates by cationic OPs were slowed down. When peripheral site ligand concentrations extended to millimolar, the extent of the enhancement decreased creating a bell shaped activation profile. Analysis of inhibition by DDVP and haloxon revealed that peripheral site inhibitors increased the second order reaction rates by increasing maximal rates of phosphylation.
Four compounds were prepared: 3-hydroxy-1-methylquinuclidinium iodide (I), 3-(N,N-dimethylcarbamoyloxy)-1-methylquinuclidinum iodide (II), and two conjugates of I and II with 2-hydroxyiminomethyl-3-methylimidazole in which two parts of the molecule were linked by -CH2-O-CH2- (III and IV). III and IV are new compounds and their synthesis and physical data were given. All compounds were tested as inhibitors of human erythrocyte acetylcholinesterase (EC 3.1.1.7, AChE). The enzyme activity was measured in 0.1 M phosphate buffer (pH 7.4) at 10 and 37 degrees C with acetylthiocholine (ATCh) as the substrate. The obtained enzyme/inhibitor dissociation constants were between 0.05 and 0.5 mM at 10 degrees C and between 0.2 and 0.6 mM at 37 degrees C. At both temperatures compounds III and IV had higher affinities for the enzyme than compounds I and II and this difference was more pronounced at 10 than at 37 degrees C. The carbamates II and IV were also progressive AChE inhibitors. For compound II the rate constants of inhibition were 6300 and 2020 M(-1) min(-1) at 37 and 10 degrees C, respectively. Compound IV was a very weak carbamoylating agent with rate constants of inhibition of 100 and 63 M(-1) min(-1) at 37 and 10 degrees C, respectively. The oxime group in compounds III and IV hydrolyzed ATCh at rates of 23 and 3.2 M(-1) min(-1) at 37 and 10 degrees C, respectively.
        
Title: Paraoxonase and arylesterase activities in the serum of two hyperlipoproteinaemic patients after repeated extracorporal lipid precipitation Reiner E, Svedruzic D, Simeon-Rudolf V, Lipovac V, Gavella M, Mrzljak V Ref: Chemico-Biological Interactions, 119-120:405, 1999 : PubMed
The effect of heparin-induced extracorporal lipid precipitation (HELP) on the activities of paraoxonase (EC 3.1.8.1) and arylesterase (EC 3.1.1.2) was studied in serum of a patient with hyperlipoproteinaemia (A) and of a patient with non-insulin dependent diabetes mellitus and hyperlipoproteinaemia (B). The enzyme activities were measured spectrophotometrically (Tris-HCl buffer, pH 7.4, 37 degrees C) with paraoxon and phenylacetate as substrates of paraoxonase and arylesterase, respectively. Both patients underwent HELP applications once a week over a period of 7 weeks. Over that period no overall change was observed either in enzyme activities or in the lipid and protein content of the sera. However, each HELP session caused an immediate decrease of EDTA-insensitive arylesterase activity (on average 56% in A and 42% in B), while EDTA-sensitive arylesterase remained almost unaltered. Paraoxonase remained unchanged in A, but decreased in B (approximately 60%). Of the atherogenic lipoprotein parameters, the most pronounced decrease was found in VLDL-cholesterol and in triglycerides (on average 45% in A and 32% in B), while the anti-atherogenic HDL-cholesterol decreased < 10%. Possible implications of the effect of HELP on the enzyme activities studied remain to be explained.
        
Title: Novel protein targets for organophosphorus compounds Richards P, Johnson M, Ray D, Walker C Ref: Chemico-Biological Interactions, 119-120:503, 1999 : PubMed
Inhibition of tritiated di-isopropyl phosphorofluoridate labelling by a range of organophopshorus compounds was used to screen for novel OP-reactive targets in rat-brain homogenates. Analysis of target proteins was conducted by SDS/PAGE and detection of tritiated proteins using a thin layer chromatography (TLC) linear analyser. Two major sites of 3H-DFP labelling were found with relative molecular masses of 30 and 85 kDa. Rates of reaction of these labelling sites with a range of OP compounds were compared to that of acetylcholinesterase. The 30 kDa band was found to be more sensitive to paraoxon, dichlorvos and diazoxon than acetylcholinesterase. The 85 kDa band was found to be more sensitive to dichlorvos and diazoxon than acetylcholinesterase. Neither labelling band reacted with chlorfenvinphos or demeton-s-methyl at significant rates.
        
Title: Identification of two rat liver proteins with paraoxonase activity: biochemical evidence for the identity of paraoxonase and arylesterase Rodrigo L, Gil F, Hernandez AF, Pla A Ref: Chemico-Biological Interactions, 119-120:263, 1999 : PubMed
The existence of two or more enzyme forms with paraoxonase activity has been reported in sheep, rabbit, human and rat serum and recently in mouse and rat liver. In this study we describe the presence of two peaks with paraoxonase activity (M1 and M2) after non-specific affinity chromatography of rat liver microsomes on Cibacron Blue 3GA. The first peak (M1) was obtained during the washing of the column and coeluted with albumin. The second active peak (M2) was eluted with 1 M NaCl. The characterization of each peak was determined by SDS/PAGE electrophoresis and Western-blotting. A comparison of both active fractions on the basis of kinetic parameters, heat inactivation and pH stability, calcium requirement and inhibition by EDTA and several metals was performed. Our results support the fact that two proteins capable of hydrolyzing paraoxon are present in rat liver microsomes. Furthermore, during the purification to homogeneity of rat liver paraoxonase we have performed a study of its hydrolytic ability against three different substrates: paraoxon, phenylacetate and phenyl thioacetate (Paraoxonase (PON), Arylesterase (ArE), Phenyl thioacetate esterase (PTase)). The elution profile in different chromatographic steps, as well as the activity ratios from the crude extract throughout the purification process, heat inactivation and effect of inhibitors were used as identity criteria for the three hydrolytic activities. Our results show evidence for the hydrolysis of paraoxon and phenylacetate by the same protein from rat liver (paraoxonase).
        
Title: A steric blockade model for inhibition of acetylcholinesterase by peripheral site ligands and substrate Rosenberry TL, Mallender WD, Thomas PJ, Szegletes T Ref: Chemico-Biological Interactions, 119-120:85, 1999 : PubMed
The active site gorge of acetylcholinesterase (AChE) contains two sites of ligand binding, an acylation site near the base of the gorge and a peripheral site at its mouth. We recently introduced a steric blockade model which demonstrated that small peripheral site ligands like propidium can inhibit substrate hydrolysis simply by decreasing the substrate association and dissociation rate constants without altering the equilibrium constant for substrate binding to the acylation site. We now employ our nonequilibrium kinetic analysis to extend this model to include blockade of the dissociation of substrate hydrolysis products by bound peripheral site ligand. We also report here that acetylthiocholine can bind to the AChE peripheral site with an equilibrium dissociation constant K(S) of about 1 mM. This value was determined from the effect of the acetylthiocholine concentration on the rate at which fasciculin associates with the peripheral site. When substrate binding to the peripheral site is incorporated into our steric blockade model, hydrolysis rates at low substrate concentration appear to be accelerated while substrate inhibition of hydrolysis occurs at high substrate concentration. The model predicts that hydrolysis rates for substrates which equilibrate with the acylation site prior to the acylation step should not be inhibited by bound peripheral site ligand. Organophosphates equilibrate with AChE prior to phosphorylating the active site serine residue, and as predicted propidium had little effect on the phosphorylation rate constants for the fluorogenic organophosphate ethylmethyl-phosphonylcoumarin (EMPC). The 2nd-order phosphorylation rate constant kOP/K(OP) was decreased 3-fold by a high concentration of propidium and the 1st-order rate constant kOP increased somewhat. In contrast to propidium, when the neurotoxin fasciculin bound to the AChE peripheral site both a steric blockade and a conformational change in the acylation site appeared to occur. With saturating fasciculin, kOP/K(OP) decreased by a factor of more than 750 and kOP decreased 300-fold. These data suggest that new peripheral site ligands may be designed to have selective effects on AChE phosphorylation.
        
Title: In vitro sequestration of two organophosphorus homologs by the rat liver Santhoshkumar P, Shivanandappa T Ref: Chemico-Biological Interactions, 119-120:277, 1999 : PubMed
Bromophos (Bp) and ethylbromophos (EBp) are two structurally homologous organophosphorus insecticides (OP) which show a 24-fold difference in their toxicity to the laboratory rat (LD50--2215 and 91 mg/kg b.w., respectively). The role of rat liver in the sequestration of the OP oxons was studied based on carboxylesterase (CaE) inhibition in vitro. Bromoxon (Bo) and ethylbromoxon (EBo) were greater inhibitors of rat hepatic CaE than brain acetylcholinesterase (AChE) with IC50 values at nanomolar and picomolar levels, respectively. The capacity of the liver to sequester OPs was determined by measuring AChE inhibition pre-incubated with or without liver homogenate. AChE inhibition by Bo decreased with increasing concentration of liver tissue, whereas it was unaffected in the case of EBo. The results imply that liver tissue contains binding sites, which sequester Bo thereby reducing the number of OP molecules available to inhibit AChE. Although CaE inhibition leads to sequestration, other binding sites in the liver may have a significant role in determining the toxicity of OPs. Differential sequestration of the OPs by hepatic tissue, therefore, could be important in understanding the role of differential saturation of the target molecules, which has a bearing on differential toxicity.
        
Title: Toxicological significance in the cleavage of esterase-beta-glucuronidase complex in liver microsomes by organophosphorus compounds Satoh T, Suzuki S, Kawai N, Nakamura T, Hosokawa M Ref: Chemico-Biological Interactions, 119-120:471, 1999 : PubMed
Egasyn is an accessory protein of beta-glucuronidase (beta-G) in the liver microsomes. Liver microsomal beta-G is stabilized within the luminal site of the microsomal vesicles by complexation with egasyn which is one of the carboxylesterase isozymes. We investigated the effects of organophosphorus compounds (OPs) such as insecticides on the dissociation of egasyn-beta-glucuronidase (EG) complex. The EG complex was easily dissociated by administration of OPs, i.e. fenitrothion, EPN, phenthionate, and bis-beta-nitrophenyl phosphate (BNPP), and resulting beta-G dissociated was released into blood, leading to the rapid and transient increase of plasma beta-G level with a concomitant decrease of liver microsomal beta-G level. In a case of phenthionate treatment, less increase in plasma beta-G level was observed, as compared with those of other OPs. This may be explained by the fact that phenthionate was easily hydrolyzed by carboxylesterase. Similarly, carbamate insecticides such as carbaryl caused rapid increase of plasma beta-G level. In contrast, no significant increase of plasma beta-G level was observed when pyrethroid insecticides were administered to rats. This is due to the fact that pyrethroids such as phenthrin and allethrin were easily hydrolyzed by A-esterase as well as carboxylesterase. On the other hand, addition of OPs to the incubation mixture containing liver microsomes caused the release of beta-G from microsomes to the medium. From these in vivo and in vitro data, it is concluded that increase of the plasma beta-G level after OP administration is much more sensitive biomarker than cholinesterase inhibition to acute intoxication of OPs and carbamates.
        
Title: Differences in active-site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase Saxena A, Redman AM, Jiang X, Lockridge O, Doctor BP Ref: Chemico-Biological Interactions, 119-120:61, 1999 : PubMed
We examined the role of A328(F330) in the binding of various inhibitors to cholinesterases (ChEs) using human butyrylcholinesterase (BChE) mutants to determine if the conclusions drawn from studies with acetylcholinesterase (AChE) mutants could be extended to BChE. For huperzine A and edrophonium, the results obtained with AChE mutants could be directly correlated with those obtained with native ChEs and site-specific mutants of human BChE. Inhibition studies of ethopropazine with BChE mutants, where A328 was modified to either F or Y, suggested that A328 was not solely responsible for the selectivity of ethopropazine. Volume calculations for the active-site gorge showed that the poor inhibitory activity of ethopropazine towards AChE was due to the smaller dimension of the active-site gorge. The volume of the BChE active-site gorge is approximately 200 A3 larger than that of the AChE gorge, which allows the accommodation of ethopropazine in two different orientations as demonstrated by rigid-body refinement and molecular dynamics calculations. These results suggest that, although the overall scaffolding of the two enzymes may be highly similar, the dimensions and the micro-environment of the gorge play a significant role in determining the selectivity of substrate and inhibitors for ChEs.
        
Title: Grafting of genetically modified human fetal fibroblasts to produce human butyrylcholinesterase in mice Shao H, Huang YZ, Wang D, Zhang H, Sun MJ Ref: Chemico-Biological Interactions, 119-120:361, 1999 : PubMed
Human diploid fibroblast cultures were established from fetal skin tissue. Enzymic dissociation yielded cultures of higher growth capacity of fibroblasts than those prepared by mechanical dissociation followed by spontaneous outgrowth of cells. Transfer of recombinant human butyrylcholinesterase (BChE, EC 3.1.1.8) gene into primary human fibroblasts was achieved successfully using lipofection and retrovirus-mediated transfection. The analysis of drug-resistant colonies suggested the presence of the transcripted BChE mRNA in the cytoplasm of transfected cells. The secreted BChE protein in culture medium was assayed for enzyme activity using butyrylthiocholine as substrate. The genetically modified fibroblasts were mixed with rat tail collagen and transplanted subcutaneously and intraperitoneally to mice. Immunoreactive human BChE appeared in the plasma from the transplanted mice. reaching the top level at day 13. It was not present any longer in most of the mice 20 days later.
Neuropathy target esterase (NTE) was shown to be an excellent biochemical marker for screening of organophosphates (OPs) with respect to their ability to result in organophosphate induced delayed neurotoxicity (OPIDN). This paper describes a new biosensor approach to the analysis of NTE and its inhibitors. The method is based on the combination of NTE enzymatic hydrolysis of phenyl valerate (PV) with phenol detection by the Clark-type oxygen electrode modified by immobilized tyrosinase. The validity of this biosensor method is confirmed by the facts that the calibration curves for NTE obtained by colorimetric and flow-through electrochemical methods were nearly identical and the titration of NTE by test inhibitor mipafox was shown to yield the same pI50 values. The developed electrochemical methods can be considered as a promising approach both for serial express NTE analysis and for kinetic characteristics of NTE.
Determination of the three dimensional structure of Torpedo Californica acetylcholinesterase (TcAChE) provided an experimental tool for directly visualizing interaction of AChE with cholinesterase inhibitors of fundamental, pharmacological and toxicological interest. The structure revealed that the active site is located near the bottom of a deep and narrow gorge lined with 14 conserved aromatic amino acids. The structure of a complex of TcAChE with the powerful 'transition state analog' inhibitor, TMTFA, suggested that its orientation in the experimentally determined structure was very similar to that proposed for the natural substrate, acetylcholine, by manual docking. The array of enzyme-ligand interactions visualized in the TMFTA complex also are expected to envelope the unstable TI that forms with acetylcholine during acylation, and to sequester it from solvent. In our most recent studies, the crystal structures of several 'aged' conjugates of TcAChE obtained with OP nerve agents have been solved and compared with that of the native enzyme. The methylphosphonylated-enzyme obtained by reaction with soman provides a useful structural analog for the TI that forms during deacylation after the reaction of TcAChE with acetylcholine. By comparing these structures, we conclude that the same 'oxyanion hole' residues, as well as the aromatic side chains constituting the 'acyl pocket', participate in acylation (TMTFA-AChE) and deacylation (OP-AChE), and that AChE can accommodate both TIs at the bottom of the gorge without major conformational movements.
        
Title: Reversible inhibition of acetylcholinesterase and butyrylcholinesterase by 4,4'-bipyridine and by a coumarin derivative Simeon-Rudolf V, Kovarik Z, Radic Z, Reiner E Ref: Chemico-Biological Interactions, 119-120:119, 1999 : PubMed
Inhibition of recombinant mouse wild type AChE (EC 3.1.1.7) and BChE (EC 3.1.1.8), and AChE peripheral site-directed mutants and human serum BChE variants by 4,4'-bipyridine (4,4'-BP) and the coumarin derivative 3-chloro-7-hydroxy-4-methylcoumarin (CHMC) was studied. The enzyme activity was measured with acetylthiocholine as substrate. Enzyme-inhibitor dissociation constants for the catalytic and peripheral sites were evaluated from the apparent dissociation constants as a function of the substrate concentration. Inhibition by 4,4'-BP of AChE, BChE and the AChE mutant Y72N/Y124Q/W286A, was consistent with inhibitor binding to both catalytic and peripheral sites. The dissociation constants for the peripheral site were about 3.5-times higher than for the catalytic site. The competition between CHMC and substrate displayed two binding sites on the AChE mutants Y72N, Y124Q, W286A and W286R, and on the atypical and fluoride-resistant BChE variants. The dissociation constants for the peripheral site were on average two-times higher than for the catalytic site. CHMC displayed binding only to the catalytic site of Y72N/Y124Q/W286A mutant and only to the peripheral site of w.t. AChE and the human usual BChE. Modelling of the 4,4'-BP and CHMC binding to wild type mouse AChE substantiated the difference between the inhibitors in their mode of binding which was revealed in the kinetic studies.
        
Title: An explanation for the different inhibitory characteristics of human serum butyrylcholinesterase phenotypes deriving from inhibition of atypical heterozygotes Simeon-Rudolf V, Kovarik Z, Skrinjaric-Spoljar M, Evans RT Ref: Chemico-Biological Interactions, 119-120:159, 1999 : PubMed
The time course of inhibition of butyrylcholinesterase (EC 3.1.1.8) by the dimethylcarbamate Ro 02-0683 in sera taken from patients heterozygous for the usual (U), atypical (A), K or J variants was followed using propionylthiocholine as substrate. Data obtained were used to determine rate constants of inhibition together with the contribution made by each variant to total enzyme activity. The findings substantiate earlier reports that J and K mutations lead to quantitative changes in the concentration of usual enzyme in contrast to the qualitative changes of the atypical variant. The contribution of the atypical enzyme to the total activity in serum from UA, AK and AJ heterozygotes was respectively 17-20, 24-31 and 34-53%. The altered ratios of atypical to usual, K or J enzyme in UA, AK and AJ together with the constants on the usual enzyme alone, explain the differences in observed inhibitor numbers which enable these heterozygotes to be identified.
        
Title: Catalytic parameters for the hydrolysis of butyrylthiocholine by human serum butyrylcholinesterase variants Simeon-Rudolf V, Reiner E, Evans RT, George PM, Potter HC Ref: Chemico-Biological Interactions, 119-120:165, 1999 : PubMed
Catalysed hydrolysis of butyrylthiocholine (BTCh) by the usual (UU), fluoride-resistant (FS), AK, AJ and atypical (AA) human serum butyrylcholinesterase (EC 3.1.1.8) variants was measured in phosphate buffer pH 7.4 at 25 degrees C. pS-curves for all phenotypes were S-shaped; the activities rose to a plateau with increasing substrate concentration except at 100 mM where there was a small decrease. To obtain the catalytic constants, three equations were applied: Michaelis-Menten equation (Eq. 1), Hill equation (Eq. 2) and an equation which assumes simultaneous binding of the substrate to the catalytic site and to a peripheral site on the enzyme (Eq. 3). Over a range from 0.01 to 50 mM BTCh, the activity versus substrate concentration relationship deviated from Michaelis-Menten kinetics (Eq. 1) while data fitted well with Eqs. 2 and 3. The Michaelis-Menten equation was applied separately to two BTCh concentration ranges: the corresponding Km constants for the UU, FS, AK, AJ and AA phenotypes ranged from 0.1 to 0.2 mM (at 0.01-1.0 mM BTCh) and from 0.3 to 2.0 mM (at 1.0-50 mM BTCh). Hill coefficients (nH) calculated from Eq. 2 were similar for all phenotypes (nH approximately 0.5). The dissociation constants K1 and K2 calculated from Eq. 3 for two sites on the enzyme fell between 0.02 and 0.12 mM (K1) and 0.89 and 4.9 mM (K2) for the five phenotypes. Experimental data support the assumption that the phenotypes studied have two substrate binding sites.
        
Title: Properties of the retained N-terminal hydrophobic leader sequence in human serum paraoxonase/arylesterase Sorenson RC, Aviram M, Bisgaier CL, Billecke S, Hsu C, La Du BN Ref: Chemico-Biological Interactions, 119-120:243, 1999 : PubMed
Human serum paraoxonase/arylesterase (PON1) is HDL-associated and appears to protect low density lipoproteins (LDL) from oxidation. Mature PON1 retains its N-terminal hydrophobic signal sequence, which may be needed for binding to HDL. By site-directed mutagenesis, we created a mutant PON1 (A19A20) with a cleavable N-terminus to determine if this peptide mediated binding to lipoproteins. As a model system, we studied binding of mutant and wild type PON1s to lipoproteins in fetal bovine serum-containing expression medium and found that the wild type recombinant enzyme associated with lipoproteins whereas the A19A20 mutant did not. These results show that the N-terminus is required for binding to either apolipoproteins or phospholipids. Furthermore, we showed that wild type enzyme can bind to phospholipids directly without apolipoproteins. To determine if lipid binding is a requirement for PON1's protection against LDL oxidation, we used a copper ion-induced oxidation system and found that the wild type enzyme and A19A20 mutant showed similar reductions in both peroxide and aldehyde formation. We conclude that PON1 depends upon its N-terminal hydrophobic peptide for its association with serum lipoproteins.
        
Title: Effects of organophosphates on cholinesterase activity and neurite regeneration in Aplysia Srivatsan M Ref: Chemico-Biological Interactions, 119-120:371, 1999 : PubMed
In Aplysia, a marine mollusc, acetylcholinesterase (AChE) is present in cholinergic and non-cholinergic neurons and in hemolymph. Aplysia hemolymph has a very high level of AChE which promotes neurite growth in primary cultures of dopaminergic neurons via a non-catalytic mechanism. In contrast, AChE is known to facilitate neurite growth in cholinoceptive neurons by hydrolyzing ACh which inhibits neurite growth. In order to test whether AChE's site-specific neurotrophic action varies with the neuronal phenotype, we investigated the effects of active-site inhibited hemolymph AChE on neurite growth of cholinergic neurons of Aplysia in primary culture. Organophosphates being long-acting active site inhibitors of AChE were chosen for this study. The effects of active site inhibited hemolymph AChE was tested on large cholinergic neurons, R2 (abdominal ganglion) and LPL1 (left pleural ganglion) as well as small cholinergic neurons (buccal ganglion) of Aplysia, maintained in culture. Partially purified hemolymph AChE was inhibited by either 10 microM of echothiophate or 5 microM of paraoxon. Neurons were maintained in (1) L15 (defined medium) alone; (2) L15 + echothiophate; (3) L-15 + paraoxon; (4) L-15 + hemolymph AChE; (5) L15 + hemolymph AChE + echothiophate; and (6) L-15 + hemolymph AChE + paraoxon. Addition of uninhibited hemolymph AChE significantly increased neurite growth of cultured neurons compared to L15 alone. In the presence of echothiophate-inhibited or praoxon-inhibited AChE, neurite growth was significantly reduced when compared to L15 + uninhibited AChE. While the presence of echothiophate by itself did not reduce survival or neurite growth when compared to L-15 alone, the presence of paraoxon by itself markedly reduced survival and neurite growth of cultured neurons. The results show that AChE's catalytic action contributes to enhance neurite growth in cholinergic neurons and the effects of paraoxon appears to differ from that of echothiophate on cholinergic neurons of Aplysia.
        
Title: Effect of tetramethylammonium, choline and edrophonium on insect acetylcholinesterase: test of a kinetic model Stojan J, Marcel V, Fournier D Ref: Chemico-Biological Interactions, 119-120:137, 1999 : PubMed
Cholinesterases display a non-Michaelian behaviour with respect to substrate concentration. With the insect enzyme, there is an activation at low substrate concentrations and an inhibition at high concentrations. Previous studies allow us to propose a kinetic model involving a secondary non-productive binding site for the substrate. Unexpectedly, this secondary site has a very high affinity for the substrate when the enzyme is free. On the contrary, when the catalytic site of the enzyme is occupied a strong decrease of this affinity was observed. Moreover, a substrate molecule bound to the peripheral site results in a global decrease of the acylation and/or the deacylation step. Kinetic studies with three reversible inhibitors, tetramethylammonium, edrophonium and choline supported the kinetic model and enable its further refinement.
        
Title: Inhibition of Drosophila acetylcholinesterase by 7-(methylethoxyphosphinyloxy)1-methyl-quinolinium iodide Stojan J, Marcel V, Fournier D Ref: Chemico-Biological Interactions, 119-120:147, 1999 : PubMed
The kinetic behaviour of Drosophila melanogaster acetylcholinesterase toward its substrate shows, in comparison with classic Michaelis-Menten kinetics, an apparent homotropic cooperative double activation-inhibition pattern. In order to construct an appropriate kinetic model and obtain further information on the mechanism of the catalytic action of this enzyme, the hydrolysis of acetylthiocholine in the absence and presence of different concentrations of synthetic quaternary methylphosphonate, 7-(methylethoxyphosphinyloxy)1-methyl-quinolinium iodide (MEPQ), was followed on a stopped-flow apparatus. The reaction at low substrate concentrations was followed until the change of the absorbance became negligible and at high concentrations only initial parts were recorded. A simultaneous analysis of the progress curves using numerical integration showed that the powerful organophosphonate inhibitor binds and compete with the substrate for the same binding sites. The results are also in accordance with the hypothesis that virtually every substrate or quasi-substrate molecule that enters into the gorge of active site is hydrolysed.
        
Title: Analysis of cholinesterase inactivation and reactivation by systematic structural modification and enantiomeric selectivity Taylor P, Wong L, Radic Z, Tsigelny I, Bruggemann R, Hosea NA, Berman HA Ref: Chemico-Biological Interactions, 119-120:3, 1999 : PubMed
We show here with a congeneric series of Rp- and Sp-alkoxymethyl phosphonothiolates of known absolute stereochemistry that chiral selectivity in their reaction with acetylcholinesterase can be described in terms of discrete orientational and steric requirements. Stereoselectivity depends on acyl pocket dimensions, which govern leaving group orientation and a productive association of the phosphonyl oxygen in the oxyanion hole. Overall geometry is consistent with a pentavalent intermediate where the attacking serine and leaving group are at apical positions. Oxime reactivation of the phosphonylated enzyme occurs through a similar associative intermediate presumably forming an oxime phosphonate. The oximes of differing structure show distinct angles of attacking the phosphate where the attack angles and access to the phosphorus are constrained in the sterically impacted gorge. Hence, efficacy of oxime reactivation is dependent on both oxime and conjugated phosphonate structures.
        
Title: Dimethylphosphorus metabolites in serum and urine of persons poisoned by malathion or thiometon Vasilic Z, Stengl B, Drevenkar V Ref: Chemico-Biological Interactions, 119-120:479, 1999 : PubMed
The urinary excretion rates of dimethyl-phosphate, -phosphorothioate and -phosphorodithioate were studied in six persons of whom four had ingested a concentrated solution of malathion and two of thiometon. The concentration decrease of single and total dimethylphosphorus metabolites was biphased, with a fast initial rate and a slow later rate. The excretion rate of total metabolites in the faster phase depended on the initial concentration in urine. At concentrations higher than 100 nmol/mg creatinine, the excretion half-times ranged from 7.5 to 15.4 h and at concentrations between 52 and 95 nmol/mg creatinine from 34.7 to 55.4 h. Non-metabolized malathion was detected only in one urine sample collected from one person immediately after hospitalization. Two persons poisoned with malathion were taken blood serum samples for the analysis of the parent pesticide and its metabolites on a daily basis after hospitalization. The parent pesticide was detectable in the serum only one day after the poisoning. The concentration of total malathion dimethylphosphorus metabolites in serum decreased very quickly within 1.5 days after hospitalization. The total metabolite elimination half-times were 4.1 and 4.7 h in the initial phase, and 53.3 and 69.3 days in the later slower elimination phase. There was no correlation between maximum concentrations of total metabolites measured in serum and/or urine on the day of admission to hospital and the initial depression of serum cholinesterase (BChE, EC 3.1.1.8) and erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7).
        
Title: NTE soluble isoforms: new perspectives for targets of neuropathy inducers and promoters Vilanova E, Escudero MA, Barril J Ref: Chemico-Biological Interactions, 119-120:525, 1999 : PubMed
Neural carboxylesterases can be discriminated by differential inhibition assays with organophosphorus compounds (OPs), paraoxon (O,O'-diethyl p-nitrophenyl phosphate) and mipafox (N,N'-diisopropyl phosphorodiamidofluoridate) being the ones used to discriminate esterases that should be either irrelevant or candidates as targets of the mechanism of induction of the organophosphorus-induced delayed polyneuropathy (OPIDP). The brain membrane-bound phenyl valerate esterase (PVase) defined by Dr Johnson in 1969 as neuropathy target esterase (NTE) and recently cloned by Dr Glynn and coworkers is termed here as particulate NTE due to its association to the membrane particulate fraction. It is considered as the target of OPIDP and is the activity measured in standard NTE assays and toxicity tests. Following the same operational criteria in the soluble fraction of sciatic nerve a paraoxon-resistant but mipafox-sensitive PVase activity was described and termed as S-NTE, with an apparent lower sensitivity to some inhibitors than particulate NTE. Two isoforms (S-NTE1 and S-NTE2) were subsequently separated by gel filtration chromatography. In a partly purified S-NTE2 preparation polypeptides were identified in western blots by labelling with S9B [1-(saligenin cyclic phospho)-9-biotinyldiaminononane], the same biotinylated OP used to label and isolate particulate NTE, but not with anti-particulate NTE antibodies. From sequential inhibition protocols, inhibitor washing-out and time course inhibition studies it is deduced that reversibility of inhibition is a new factor introducing a higher complexity in the identification of the esterases that could be candidates as targets of the mechanisms of induction and/or promotion of neuropathy. We have evidences that in sciatic nerve soluble fraction a high proportion (about 70%) of the activity that is inhibited by paraoxon in the usual concurrent assay is quickly reactivated after removing paraoxon and it is permanently inhibited by mipafox. Under this improved sequential paraoxon/mipafox inhibition procedure S-NTE represents about 50% of total PVases while in the usual concurrent assay it was only apparently about 1-2%. Moreover with such criteria, S-NTE2 isoform(s) represents about 97-99% of total S-NTE, and S-NTE1 is only a marginal amount probably resulting of a partial solubilization from particulate NTE. Fixed time inhibiton curves with variable mipafox concentration failed to discriminate more than one component. However kinetic behaviour of the time progressive inhibition cannot be explained by a simple model with a single exponential mathematical component, indicating that either the possibility of more than one component or a more complex mechanistic model should be considered. Consequently both particulate NTE and S-NTE assay protocols and their role in induction and promotion of neuropathies will need to be reviewed. Data published by Drs Lotti, Moretto and coworkers suggest that particulate NTE cannot be the target of promotion of axonopathies. The proposal that S-NTE2 could be such a target is suggestive and under collaborative biochemical and toxicological studies.
        
Title: Combined effects of glucocorticoids and electromechanical activity on the acetylcholinesterase expression in the fast rat muscle Vodiskar J, Zgonc V, Ribaric S, Grubic Z Ref: Chemico-Biological Interactions, 119-120:333, 1999 : PubMed
Protein synthesis is impaired in the glucocorticoid (GC)-treated fast mammalian muscle. Electromechanical activity was reported to alleviate this effect. Acetylcholinesterase (AChE; EC 3.1.1.7) synthesis in the skeletal muscle is regulated by both, GCs and electromechanical activity. In light of the above reports, one would expect that electrical stimulation will prevent GC-mediated fall of AChE synthesis in the muscle. On the other hand, a substantial body of evidence suggests that electromechanical activity exerts its effect at the AChE mRNA level, while GCs most probably act at the translational or early posttranslational level. Different levels of action would be more consistent with the independent and therefore additive influences of the two regulatory factors. In order to ascertain whether glucocorticoid and electromechanical effects interact in the control of AChE activity, we compared the effects of GCs on normal, nonstimulated fast rat skeletal muscle, with those of GC-treated and simultaneously electrically stimulated (tonic pattern, 10 Hz) muscle. Untreated and stimulated-only muscles were used as respective controls. The effects on the fast extensor digitorum longus muscle and slow soleus muscle, treated similarly were compared. As expected, chronic GC treatment and electrical stimulation of fast rat muscles with slow activity patterns both downregulated AChE activity. However, no additional decrease in AChE activity was observed, if stimulated fast muscle was simultaneously treated with GCs, suggesting that slow pattern of electromechanical activity prevents GC-mediated downregulation of AChE. The most plausible explanation of this observation is, that muscle activity blocks expression of some generally acting factors, which are induced by GCs and are responsible for the impaired synthesis of several proteins including AChE.
        
Title: Glucocorticoids differentially control synthesis of acetylcholinesterase and butyrylcholinesterase in rat liver and brain Weber U, Brank M, Grubic Z Ref: Chemico-Biological Interactions, 119-120:341, 1999 : PubMed
Mammalian organisms possess two cholinesterases: acetylcholinesterase (AChE, EC 3.1.1.7.) and butyrylcholinesterase (BuChE, EC 3.1.1.8.). A clear explanation for this dual expression of acetylcholine-hydrolyzing enzymes is still missing. Better knowledge on how these two enzymes respond to various physiological or pharmacological factors would importantly contribute to the understanding of their function. The aim of the present study is to elucidate glucocorticoid (GC) influences on the synthesis of AChE and BuChE in rat liver and brain. Female Wistar rats were treated with dexamethasone until body weight loss was greater than 15%, signaling full expression of a GC response. At this stage, liver and brain were isolated and AChE and BuChE activities were determined in their homogenates. A new approach, based on precise radiometric measurements of AChE and BuChE activities in the polysomal fractions, prepared under non-denaturing conditions, was used to study GC influences on the early stages of biosynthesis of both enzymes. We found a differential GC influence on AChE and BuChE. In brain, only BuChE activity was affected (-30%), while AChE remained practically unchanged. In liver, BuChE activity fell by 60%, while AChE lost only 18% of its control activity. In case of BuChE, decreased activities in the whole homogenates correlated with decreased activities in the polysomal fractions, suggesting that early stages of enzyme biosynthesis were primarily affected. On the other hand, decreased AChE activity in liver homogenates was not paralleled by a significant change at the level of polysomal AChE activity in this organ, suggesting that higher AChE turn-over is primarily responsible for the decreased activity in homogenate. These results, together with the GC-mediated elimination of the correlation between brain and liver BuChE activities, strongly support the proposal of Edwards and Brimijoin (J.A. Edwards, S. Brimijoin, Effects of hypophysectomy on acetylcholinesterase and butyrylcholinesterase in the rat, Biochem. Pharmacol. 32 (1983) 1183-1189) that BuChE is regulated by systemically acting factors, including various hormones, while regulation of AChE is primarily tissue-specific.
One allele of the AChE gene (ACHE) was knocked out in embryonic stem (ES) cells by homologous recombination. The targeting vector contained 2 kb of a TK gene cassette for negative selection, 884 bp of ACHE including exon 1, 1.6 kb of a Neo(r) gene cassette for positive selection, 5.2 kb of the ACHE Bam HI fragment including exon 6, and 3 kb of Bluescript. The use of this vector deleted exons 2-5, which removed 93% of the ACHE coding sequence including the signal peptide, the active site serine, and the histidine and glutamic acid of the catalytic triad. The gene targeting vector was transfected into ES cells by electroporation. Colonies resistant to G418 and gancyclovir were screened for homologous recombination by Southern blotting. Out of 200 colonies, four were found to have undergone homologous recombination. These four ACHE (+/-) ES cell lines were expanded to provide cells for microinjection into C57Bl/6 mouse blastocysts. The injected blastocysts were implanted into pseudopregnant CD/l white mice. More than 200 injected blastocysts were transferred into 20 mice. More than 65 mice were born, of which 11 were chimeras. Chimeras were identified by their black and agouti coat color. Littermates were all black. Thus far, seven male chimeras have been bred with more than 130 C57Bl/6 females to generate 26 agouti mice out of 199 living offspring. This demonstrated that the ACHE (+/-) ES cells contributed to the germline. Offspring with agouti coat color have a 50% chance of carrying the knockout allele. The 26 agouti offspring were screened for an ACHE (+/-) genotype by tail biopsy PCR. Ten out of 26 agouti mice are heterozygous ACHE knockout mice, and they are healthy and alive at 29 days of age. We expect a phenotype to appear in nullizygous animals.
Paraoxonase in serum and liver of rabbits and cattle was investigated. In serum the two substrates paraoxon and phenylacetate are exclusively hydrolyzed by alpha-lipoprotein-bound paraoxonase. In rabbit liver paraoxon is hydrolyzed only by paraoxonase, while phenylacetate is hydrolyzed by paraoxonase (20%) and additionally by an organophosphate sensitive carboxylesterase (B-Esterase), which is responsible for 80% of total liver phenylacetate hydrolysis. Phenyl acetate hydrolysis by B-Esterase of rabbit liver was shown to be inhibited by paraoxon and by mipafox covalently in a time and concentration dependent manner. Rabbit serum exhibits by far the highest serum paraoxonase activity (2.6 +/- 0.66 U/ml) of all vertebrate species tested up to now, while rabbit liver contains only 0.5 +/- 0.2 U/g fresh weight. In cattle extremely high paraoxonase activity is found in liver (2.8 U/g), while bovine serum contains only 0.2 U/g. The paraoxonase activity ratio (hydrolysis rate paraoxon: phenylacetate x 1000) in cattle does not show interindividual variation (activity ratio 4.0 +/- 0.4, correlation coefficient 0.996, P < 0.001). In contrast, the paraoxon/phenylacetate hydrolysis ratio of rabbit paraoxonase in serum as well as in liver does vary considerably between individuals. In cross-bred rabbits paraoxonase activity ratios from three to ten are found. In a strain of pure-bred New Zealand White rabbits three polymorphic serum paraoxonase phenotypes could be clearly differentiated by the activity ratio. By analogy with the human paraoxonase polymorphism, the rabbit paraoxonase isotypes were classified as paraoxonase A (activity ratio 3.8-4.3), AB (ratio 5.5-6.0) and B (ratio 7.3-8.6). The corresponding frequencies of the three isotypes were 40, 35 and 25%.