Genetic polymorphism of eserine resistant esterases in canine plasma

Six plasma eserine resistant esterase phenotypes were observed in a population of 1438 dogs consisting of 38 breeds. Analysis of parentage records of the dogs examined revealed that the phenotypic variation of eserine resistant esterases was controlled by 3 codominant alleles ESA, ESB and ESC at one autosomal locus. The gene frequency of ESB was high in most of the breeds examined. Allele ESC was only seen in 5 Japanese native breeds, Akita, Shikoku, Hokkaido, Shinshu-Shiba and Mino-Shiba, and in a Spitz dog. Allele ESA has a low frequency in Japanese breeds but a higher frequency in some of the European breeds tested.     

The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.     

Inhibition of spicule elongation in sea urchin embryos by the acetylcholinesterase inhibitor eserine

The activity of acetylcholinesterase (AchE) increases rapidly after the gastrula stage of sea urchin development. In this report, changes in activity and in the molecular differentiation of AchE were investigated. AchE activity increased slightly during gastrulation and rose sharply thereafter, and was dependent on new RNA synthesis. No activity of butyrylcholinesterase was found. Morphogenesis in sea urchin embryos was inhibited by the AchE inhibitor eserine, which specifically inhibited arm rod formation but not body rod formation. Spicule formation and enzyme activity in cultured micromeres were inhibited by eserine in a dose-dependent manner. During gastrulation, two molecular forms of AchE were detected with polyacrylamide gel electrophoresis. The appearance of an additional band on the gel was consistent with the occurrence of a remarkable increase in the enzyme activity. This additional band appeared as a larger molecular form in Anthocidaris crassispina, Hemicentrotus pulcherrimus, Stomopneustes variolaris, and Strongylocentrotus nudus, and as a smaller form in Clypeaster japonicus and Temnopleurus hardwicki. These results suggest that the change in the molecular form of AchE induced a change in enzymatic activity that in turn may play a role in spicule elongation in sea urchin embryos.     

棉铃虫AChE不同分子型对抑制剂的敏感度及其与抗药性的关系(英文)

在分离到的棉铃虫AChE五种不同的分子型中 ,2 .1s和 8.7sAChE抗性品系对毒扁豆碱的敏感度明显低于敏感品系 ,成虫头部I50 值分别相差 1 86.3和 84.8倍 ,幼虫I50 值分别相差 1 0 1 0 倍和 1 0 5 倍。幼虫 5.3sAChE对毒扁豆碱的敏感度抗性品系和敏感品系相差达 1 2 3倍 ,而成虫则没有差异。研究结果表明 2 .1s、5.3s和 8.7sAChE敏感度降低可能是造成棉铃虫对有机磷和氨基甲酸酯类杀虫药剂产生抗性的主要原因     

Genetic polymorphism of eserine resistant esterases in canine plasma*

Six plasma eserine resistant esterase phenotypes were observed in a population of 1438 dogs consisting of 38 breeds. Analysis of parentage records of the dogs examined revealed that the phenotypic variation of eserine resistant esterases was controlled by 3 codominant alleles Es^A, Es^Band Es^Cat one autosomal locus. The gene frequency of Es^Bwas high in most of the breeds examined. Allele Es^Cwas only seen in 5 Japanese native breeds, Akita, Shikoku, Hokkaido, Shinshu-Shiba and Mino-Shiba, and in a Spitz dog. Allele Es^Ahas a low frequency in Japanese breeds but a higher frequency in some of the European breeds tested.     

Mutations of acetylcholinesterase1 contribute to prothiofos-resistance in Plutella xylostella (L.)

Insensitive acetylcholinesterase (AChE) is involved in the resistance of organophosphorous and carbamate insecticides. We cloned a novel full-length AChE cDNA encoding ace1 gene from adult heads of the diamondback moth (DBM, Plutella xylostella). The ace1 gene encoding 679 amino acids has conserved motifs including catalytic triad, choline-binding site and acyl pocket. Northern blot analysis revealed that the ace1 gene was expressed much higher than the ace2 in all examined body parts. The biochemical properties of expressed AChEs showed substrate specificity for acetylthiocholine iodide and inhibitor specificity for BW284C51 and eserine. Three mutations of AChE1 (D229G, A298S, and G324A) were identified in the prothiofos-resistant strain, two of which (A298S and G324A) were expected to be involved in the prothiofos-resistance through three-dimensional modeling. In vitro functional expression of AChEs in Sf9 cells revealed that only resistant AChE1 is less inhibited with paraoxon, suggesting that resistant AChE1 is responsible for prothiofos-resistance.     

取食不同寄主植物对棉蚜后代抗药性的影响

测定了5种药剂对棉蚜Aphis gossypii抗氰戊菊酯、吡虫啉品系和敏感品系取食棉花、黄瓜和石榴的后代的毒力,并对它们的后代体内乙酰胆碱酯酶和羧酸酯酶的比活力做了初步探索。结果表明,氰戊菊酯抗性品系取食棉花比取食黄瓜的后代对氰戊菊酯的抗性大76.4倍,对灭多威、氧乐果、硫丹和吡虫啉的抗性也大0.5～4.6倍;取食石榴的后代对5种药剂的抗性介于取食棉花和黄瓜的之间。吡虫啉抗性品系的测定结果与氰戊菊酯抗性品系基本一致。敏感品系取食黄瓜比取食棉花的后代对5种药剂的敏感性更高。3个品系取食不同植物的后代相比,其体内乙酰胆碱酯酶的比活力,取食棉花的为取食黄瓜的2.4～2.8倍;羧酸酯酶的比活力,取食棉花的为取食黄瓜的1.8～2.4倍。证明棉蚜的抗性和敏感品系取食的寄主植物不同,可引起对药剂敏感性的变化。乙酰胆碱酯酶和羧酸酯酶活力的变化均是引起这种变化的重要因素。     

A COMPARISON OF SENSITIVITY TO INHIBITOR AMONG ACETYLCHOLINESTERASE (AChE) MOLECULAR FORMS OF RESISTANT AND SUSCEPTIBLE STRAINS IN HELICOVERPA ARMIGERA*

Abstract The sensitivity of 2.8s and 8.7s acetylcholinesterase (AChE) to eserine sulfate is significantly lower in resistant (R) strain than in susceptible (S) strain in five AChE forms isolated by sucrose gradient centrifugation from cotton bollworm, Helicoverpa armigera. There are 186 and 85 times of difference in heads of adults and 10¹⁰ and 10⁵ times of difference in heads of larvae based on a comparison of I₅₀ values for 2.8s and 8.7s forms respectively. The sensitivity of 5.3s form of AChE to eserine sulfate shows 123 times of difference between R and S strains in larvae, however no difference in adults. The above results indicate that insensitive 2.8s, 8.7s and 5.3s forms of AChE may play an important role in the resistance of cotton boll‐worm to organophosphate and carbamate insecticides.     

The genetic basis of malathion resistance in housefly (Musca domestica L.) strains from Turkey

Organophosphate insecticide (parathion/diazinon) resistance in housefly (Musca domestica L.) is associated with the change in carboxylesterase activity. The product of MdalphaE7 gene is probably playing a role in detoxification of xenebiotic esters. In our research, we have isolated, cloned and sequenced the MdalphaE7 gene from 5 different Turkish housefly strains. High doses of malathion (600 microg/fly) were applied in a laboratory environment for one year to Ceyhan1, Ceyhan2, Adana and Ankara strains while no insecticide treatment was performed in the laboratory to Kirazli strain. Trp251 --> Ser substitution was found in the product of MdalphaE7 gene in all malathion resistant and Kirazli stocks. In addition, we checked the malathion carboxylesterase (MCE), percent remaining activities in acetylcholinesterase (AChE), glutathion-S-transferase (GST), and general esterase activities in all 5 strains used in this study. In comparing with universal standard sensitive control WHO, a high level of MCE and GST activities were observed while lower level of general esterase activities was detected in the tested strains. In addition, a higher percent remaining activities in AChE than WHO susceptible strain were observed in all malathion resistant strains.     

Biphasic Effect of Eserine and Other Acetylcholinesterase Inhibitors on the Nicotinic Response to Acetylcholine in Cultured Adrenal Chromaffin Cells

Abstract: A pharmacological study was made of the effects of various anticholinesterases (anti-ChEs) on the release of [³H]noradrenaline ([³H]NA) evoked by acetylcholine (ACh), nicotine, 56 mM K⁺, and veratridine from bovine adrenal chromaffin cells in culture. The anti-ChEs chosen were eserine (an inhibitor of both acetylcholinesterase and pseudocholinesterase), 1,5-bis-(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284C51) (a specific acetylcholinesterase (AChE) inhibitor), and tetraisopropylprophos-phoramide (iso-OMPA) (a specific pseudocholinesterase inhibitor). Acetylcholinesterase (AChE) activity increased in the cells with time in culture beginning at day 4 and reaching a plateau at day 10. In 9–11-day cultures, both eserine and BW284C51 acted biphasically to increase ACh-induced [³H]NA release from the cells at concentrations of 10^?6M or less whereas higher concentrations reduced or abolished the ACh-induced release. However, in earlier cultures (days 3–5), when AChE activity of the cells was low, both eserine and BW284C51 produced only a monophasic dose-dependent inhibition of ACh-evoked [³H]NA release at high concentrations. When the cells were stimulated with nicotine, an agonist not metabolized by cholinesterase a similar monophasic inhibitory response on the [³H]NA release was elicited by eserine and BW284C51, regardless of the age of the cultured cells. When 56 mM K⁺ or veratridine was used to depolarize the cells, neither eserine nor BW284C51 affected the [³H]NA release from the cells. Unlike eserine and BW284C51, iso-OMPA did not enhance ACh-evoked release in older cultures and at high concentrations (> 10 ⁴M) it produced an inhibition of the [³H]NA release evoked by ACh, nicotine, 56 mM K⁺, and veratridine. The present results suggest that the stimulatory effect on ACh response by low concentrations of eserine and BW284C51 can be attributed to the protection of ACh against enzymatic hydrolysis, whereas the inhibitory effects produced by higher concentrations of eserine and BW284C51 are thought to be due to an interaction with the nicotinic acetylcholine receptor-ionophore complex.     

Cholinesterase inhibitory potential of different Alternaria spp. and their phylogenetic relationships

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors are used for the treatment of various disorders related to decline in acetylcholine levels in the brain by inhibiting the activity of the neurotransmitter AChE. The present study reports the potential of endophytic Alternaria spp. for their potential to produce cholinesterase inhibitors active against both acetylcholine and butyrylcholine. Twenty-nine isolates belonging to Alternaria spp. were isolated from different plants and screened. Variation with respect to inhibitor production was observed in different isolates. Out of 29 cultures screened, good cholinesterase (both AChE and BChE) inhibitory activity in range of 70–85% was observed in three isolates, whereas three showed only AChE inhibition. No correlation was observed in AChE and BChE inhibitor production. TLC bioautography for the inhibitor in the selected cultures evinced different Rf values of inhibitors indicating different nature of the compounds produced. In order to analyze evolutionary relationships between producer and non-producer strains, phylogenetic analysis of six producer and five non-producer strains was carried out using amplified ITS-I-5.8SrDNA-ITS-II region. Phylogenetic analysis revealed majority of the non-producer strains to be present on different clades indicating different evolutionary origins. The dual cholinesterase inhibitory activity and the diversity in the inhibitors produced by different isolates could prove to be novel sources of pharmaceutical as well as agriculturally important biomolecules after purification and characterization.     

Acetylcholinesterase-ISFET based system for the detection of acetylcholine and acetylcholinesterase inhibitors

A bioelectronic hybrid system for the detection of acetylcholine esterase (AChE) catalytic activity was assembled by way of immobilizing the enzyme to the gate surface of an ion-sensitive field-effect transistor (ISFET). Photometric methods used to characterize bonded enzyme and linker layers on silicon substrates confirm the existence of a stable amino-cyanurate containing AChE monolayer. The transduction of the enzyme-functionalized ISFET, in ionic solutions, is detected in response to application of acetylcholine (ACh). Recorded sensitivity of the modified ISFET to ACh has reached levels of up to 10(-5)M. The Michaelis-Menten constant of the immobilized AChE is only moderately altered. Nevertheless, the maximum reaction velocity is reduced by over an order of magnitude. The ISFET response time to bath or ionophoretic application of ACh from a micropipette was in the range of a second. The catalytic activity of the immobilized AChE is inhibited in a reversible manner by eserine, a competitive inhibitor of AChE. We conclude that the immobilized enzyme maintains its pharmacological properties, and thus the described bioelectronic hybrid can serve as a detector for reagents that inhibit AChE activity.     

Comparison of two acetylcholinesterase gene cDNAs of the lesser mealworm, Alphitobius diaperinus, in insecticide susceptible and resistant strains

Two cDNAs encoding different acetylcholinesterase (AChE) genes (AdAce1 and AdAce2) were sequenced and analyzed from the lesser mealworm, Alphitobius diaperinus. Both AdAce1 and AdAce2 were highly similar (95 and 93% amino acid identity, respectively) with the Ace genes of Tribolium castaneum. Both AdAce1 and AdAce2 have the conserved residues characteristic of AChE (catalytic triad, intra-disulfide bonds, and so on). Partial cDNA sequences of the Alphitobius Ace genes were compared between two tetrachlorvinphos resistant (Kennebec and Waycross) and one susceptible strain of beetles. Several single nucleotide polymorphisms (SNPs) were detected, but only one non-synonymous mutation was found (A271S in AdAce2). No SNPs were exclusively found in the resistant strains, the A271S mutation does not correspond to any mutations previously reported to alter sensitivity of AChE to organophosphates or carbamates, and the A271S was found only as a heterozygote in one individual from one of the resistant A. diaperinus strains. This suggests that tetrachlorvinphos resistance in the Kennebec and Waycross strains of A. diaperinus is not due to mutations in either AChE gene. The sequences of AdAce1 and AdAce2 provide new information about the evolution of these important genes in insects.     

Recombinant expression and biochemical characterization of the catalytic domain of acetylcholinesterase-1 from the African malaria mosquito, Anopheles gambiae

Acetylcholinesterases (AChEs) and their genes from susceptible and resistant insects have been extensively studied to understand the molecular basis of target site insensitivity. Due to the existence of other resistance mechanisms, however, it can be problematic to correlate directly a mutation with the resistant phenotype. An alternative approach involves recombinant expression and characterization of highly purified wild-type and mutant AChEs, which serves as a reliable platform for studying structure–function relationships. We expressed the catalytic domain of Anopheles gambiae AChE1 (r-AgAChE1) using the baculovirus system and purified it 2,500-fold from the conditioned medium to near homogeneity. While K_M's of r-AgAChE1 were comparable for ATC, AβMTC, PTC, and BTC, V_max's were substantially different. The IC₅₀'s for eserine, carbaryl, paraoxon, BW284C51, malaoxon, and ethopropazine were 8.3, 72.5, 83.6, 199, 328, and 6.59 × 10⁴ nM, respectively. We determined kinetic constants for inhibition of r-AgAChE1 by four of these compounds. The enzyme bound eserine or paraoxon stronger than carbaryl or malaoxon. Because the covalent modification of r-AgAChE1 by eserine occurred faster than that by the other compounds, eserine is more potent than paraoxon, carbaryl, and malaoxon. Furthermore, we found that choline inhibited r-AgAChE1, a phenomenon related to the enzyme activity decrease at high concentrations of acetylcholine.     

Kinetic characterization of all steps of the interaction between acetylcholinesterase and eserine

The three-step carbamylenzyme mechanism of the action of eserine on acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) has been known for a long time, but its complete kinetic characterization has never been done. Some of our investigations indicated that the determination of missing kinetic parameters should include the inspection of the enzyme-eserine interaction in a very wide range of eserine concentrations. Therefore, the activity of acetylcholinesterase as a function of time in the presence of low concentrations of eserine comparable to the enzyme concentration was followed. The reaction mechanism was analysed by fitting numerically integrated differential equations that describe the time dependences of all reactants and reaction intermediates to these data. Additionally, the progress curve measurements at higher eserine concentrations were carried out on a stopped-flow apparatus. The corresponding progress curve equations were derived and the kinetic parameters evaluated by non-linear regression treatment. The complex analysis confirmed the three-step mechanism. The values of the constants showed that the very high affinity of eserine for binding into the active centre of the enzyme is not so much a consequence of the fast initial complex formation but rather a consequence of its slow dissociation. The subsequent covalent bonding of eserine is also slow, but faster than the dissociation of the initial complex. In this manner, the decarbamoylation is the only process responsible for the reactivation of acetylcholinesterase after removal of eserine.     

Purification and characterization of acetylcholinesterase from cotton aphid (Aphis gossypii Glover)

A simple and effective method was set up to purify acetylcholinesterase (AChE, EC3.1.1.7) from the cotton aphid, Aphis gossypii Glover. The procedure involved filtration on a sephadex G-25 column, separation with sephadex G-200 and procainamide affinity column. AChE from both susceptible and resistant strains were purified to a single band as resolved on denaturing polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity increased by 35,100- and 33,680-fold with a yield of 30.3 and 29.8%, respectively. The molecular mass of the purified AChE was about 63,500 Dalton as determined by SDS-PAGE. However, three bands resolved on PAGE gel electrophoresis, leading to the inference that native AChE exists in three forms. The optimum conditions for measuring the activity of purified AChE with kinetic method were 0.02M phosphate buffer, pH7.2, 0.02 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and 25 degrees C. Investigation also revealed that crude extract and purified AChE had different kinetic characteristics and inhibitory properties. They responded differently to varied DTNB, ATChI, and phosphate buffer ion concentrations, as well as pH, temperature, and inhibitors. The purified AChE was more sensitive to eserine, methamidophos, and pirimicarb. Especially for resistant aphids, the sensitivity of purified AChE to methamidophos and pirimicarb was enhanced 6.43 and 11.73 times, respectively. We infer that one or more factors in the crude extract from the resistance strain have more influence on AChE sensitivity. Further study is needed to investigate the basis of these observations.     

Organophosphate-resistant forms of acetylcholinesterases in two scallops--the Antarctic Adamussium colbecki and the Mediterranean Pecten jacobaeus

We describe the acetylcholinesterase polymorphisms of two bivalve molluscs, Adamussium colbecki and Pecten jacobaeus. The research was aimed to point out differences in the expression of pesticide-resistant acetylcholinesterase forms in organisms living in different ecosystems such as the Ross Sea (Antarctica) and the Mediterranean Sea. In A. colbecki, distinct acetylcholinesterase molecular forms were purified and characterized from spontaneously soluble, low-salt-soluble and low-salt-Triton extracts from adductor muscle and gills. They consist of two non-amphiphilic acetylcholinesterases (G(2), G(4)) and an amphiphilic-phosphatidylinositol-membrane-anchored form (G(2)); a further amphiphilic-low-salt-soluble G(2) acetylcholinesterase was found only in adductor muscle. In the corresponding tissues of P. jacobaeus, we found a non-amphiphilic G(4) and an amphiphilic G(2) acetylcholinesterase; amphiphilic-low-salt-soluble acetylcholinesterases (G(2)) are completely lacking. Such results are related with differences in cell membrane lipid compositions. In both scallops, all non-amphiphilic AChEs are resistant to used pesticides. Differently, the adductor muscle amphiphilic forms are resistant to carbamate eserine and organophosphate diisopropylfluorophosphate, but sensitive to organophoshate azamethiphos. In the gills of P. jacobaeus, amphiphilic G(2) forms are sensitive to all three pesticides, while the corresponding forms of A. colbecki are sensitive to eserine and diisopropylfluorophosphate, but resistant to azamethiphos. Results indicate that organophosphate and/or carbamate resistant AChE forms are present in species living in far different and far away environments. The possibility that these AChE forms could have ensued from a common origin and have been spread globally by migration is discussed.     

抗性品系棉蚜乙酰胆碱酯酶和羧酸酯酶的变异

用浸叶法测定了采自我国不同地区(泰安、莱阳、南京、北京和安阳)的棉蚜品系Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ对久效磷、甲胺磷、抗蚜威和灭多威等杀虫剂的抗性水平,各棉蚜品系对杀虫剂的抗性依次为Ⅴ>Ⅳ>Ⅲ,Ⅱ>Ⅰ。进一步研究表明,Ⅴ和Ⅳ品系棉蚜乙酰胆碱酯酶对杀虫剂的敏感性显著下降,Ⅱ品系次之,Ⅲ和Ⅰ品系接近于敏感品系。Ⅴ和Ⅳ品系乙酰胆碱酯酶的Km值显著下降,表明酶发生了质的变化。不同棉蚜抗性品系的酯酶（全酯酶和羧酸酯酶）活性均显著升高,其中Ⅲ品系的酯酶活力为Ⅱ品系的2倍。Ⅴ品系羧酸酯酶Km值达2460.4 μmol/L,而Ⅳ品系仅为84.4 μmol/L,该两个品系羧酸酯酶发生了质的变化。研究结果表明,不同抗性程度的棉蚜品系均存在代谢抗性和靶标抗性。低抗水平的棉蚜品系,以代谢抗性为主,靶标抗性为辅;中抗水平的棉蚜品系,抑或由于解毒代谢酶的活性显著增强,也可能由于靶标的敏感性显著下降;而高抗水平的棉蚜品系,依赖于代谢抗性和靶标抗性的联合作用。     

Direct detection of acetylcholinesterase inhibitor binding with an enzyme-based surface plasmon resonance sensor

Acetylcholinesterase (AChE) inhibitors are potentially lethal but also have applications as therapeutic drugs for neurodegenerative diseases such as Alzheimer’s. Enzyme inhibitor binding are difficult to be detected directly by surface plasmon resonance (SPR) due to their small molecular weight. In this article, we describe the detection of AChE inhibitor binding by SPR without the use of competitive binding or antibodies. AChE was immobilized on the gold surface of an SPR sensor through covalent attachment to a self-assembled monolayer (SAM) of a COOH-terminated alkanethiol. The activity of the immobilized protein and the surface density were determined by using a standard photometric assay. Binding of two reversible inhibitors, which are used as therapeutic drugs, was detectable by SPR without the need to further modify the surface or the use of other reagents. The binding affinities (K_A) obtained from the fits were 3.8 × 10³ M⁻¹ for neostigmine and 1.7 × 10³ M⁻¹ for eserine, showing a higher affinity of the sensor for neostigmine. We believe that the SPR sensor’s ability to detect these inhibitors is due to conformational changes of the enzyme structure on inhibitor binding.