Heterologous expression,purification, and biochemical characterization of a greenbug (Schizaphis graminum) acetylcholinesterase encoded by a paralogous gene (ace‐1)

Acetylcholinesterase is a critical enzyme in the regulation of cholinergic neurotransmission in insects. To produce Schizaphis graminum acetylcholinesterase‐1 for structure–function analysis, we constructed a recombinant baculovirus to infect Sf9 cells, which secreted the soluble protein at a final concentration of 4.0 mg/L. The purified enzyme had an apparent M_r of 70 and 130 kDa in the reducing and nonreducing SDS‐polyacrylamide gels, respectively, indicating that it formed a dimer via an intermolecular disulfide bond. The fresh enzyme had a specific activity of 245 U/mg, which stabilized at a lower level (115 U/mg) in storage. The Michaelis constant and maximum velocity were 88.3 ± 9.6 μM and 133.2 ± 1.6 U/mg for acetylthiocholine iodide, 113.9 ± 12.5 μM and 106.4 ± 3.0 U/mg for acetyl(β‐methyl)thiocholine iodide, 68.9 ± 7.8 μM and 76.7 ± 1.0 U/mg for propionylthiocholine iodide, and 201.1 ± 21.0 μM and 4.4 ± 0.1 U/mg for S‐butyrylthiocholine iodide, respectively. The IC₅₀ values (5 min, room temperature) of ethopropazine, BW284C51, carbaryl, eserine, malaoxon, and paraoxon were 102, 1.66, 0.94, 0.20, 0.061, 0.016 μM, respectively. The bimolecular reaction constants (k_i) were (6.50 ± 0.40) × 10⁴ for carbaryl, (1.00 ± 0.16) × 10⁵ for eserine, (4.70 ± 0.13) × 10⁵ for malaoxon, and (9.06 ± 0.23) × 10⁵ M^?1 min^?1 for paraoxon. The enzyme was also inhibited by one of its products, choline, at concentrations higher than 20 mM, suggesting that choline bound to an anionic site and regulated the enzymatic activity. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:51–59, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20311     

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.     

Purification and kinetic analysis of acetylcholinesterase from western corn rootworm,Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae)

Acetylcholinesterase (AchE, EC 3.1.1.7) was purified from western corn rootworm (WCR, Diabrotica virgifera virgifera) beetles by affinity chromatography. The purification factor reached over 20,000-fold with a specific activity of 169.5 μmol/min/mg and a yield of 23%. The V_max values for hydrolyzing acetylthiocholine (ATC), acetyl-(β-methyl)thiocholine (AβMTC), propionylthiocholine (PTC), and S-butyrylthiocholine (BTC) were 184.8, 140.5, 150.2, and 18.8 μmol/min/mg, respectively, and K_m values were 19.7, 18.5, 14.1, and 11.0 μM, respectively. The first three substrates showed significant inhibition to the AchE at higher concentrations, whereas BTC showed inhibition at the concentrations of 0.25–2 mM but activation at >4 mM. AchE activity was almost completely inhibited by 1 μM eserine and BW284C15, respectively, but only 12% of AchE activity were inhibited by ethopropazine at the same concentration. These results suggested that the purified AchE from WCR was a typical insect AchE. Insecticides or their oxidative metabolites, chlorpyrifos-methyl oxon, carbofuran, carbaryl, malaoxon, and paraoxon, used in in vitro kinetic study exhibited high inhibition to AchE purified from WCR. However, chlorpyrifos-methyl oxon and carbofuran showed at least 36- and 4-fold, respectively, higher inhibitory potency than the remaining insecticides examined. Results from our in vitro inhibition of AchE agreed quite well with the previously published in vivo bioassay data. Arch. Insect Biochem. Physiol. 39:118–125, 1998. © 1998 Wiley-Liss, Inc.     

Polymorphism in the acetylcholinesterase gene of Musca domestica L. field populations in Turkey

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphates (OPs) and carbamates (CBs) in insects. Ace mutations have been identified in OP and CB resistant strains of Musca domestica. In this study, the Ace gene was partially amplified and sequenced at amino acid positions 260, 342, and 407 to determine the frequencies of these mutations in housefly samples collected from farms and garbage disposal sites of 16 provinces in the Aegean and Mediterranean regions of Turkey. In addition, the percent remaining AChE activities in these samples were assayed by using three OPs (malaoxon, paraoxon, and dichlorvos) and one CB (carbaryl) compound as inhibitors. In all the analyzed samples, 13 different combinations at the three amino acid positions were identified and the L/V260-A/G342-F/Y407 combination was found in the highest frequency. No susceptible individual was detected. The highest mean percent remaining AChE activities were detected in the individuals having the L260-A/G342-F/Y407 genotype when malaoxon and paraoxon were used as inhibitors and in the individuals with the L260-A342-F/Y407 combination when dichlorvos and carbaryl were used as inhibitors. The obtained data were heterogeneous and there was no exact correlation between the molecular genetic background and the resistance phenotypes of the flies. The findings of this study at the molecular and biochemical levels indicate the presence of significant control problems in the field.     

Comparative inhibition kinetics for acetylcholinesterases extracted from organophosphate resistant and susceptible strains of Boophilus microplus (Acari: Ixodidae)

In this study, acetylcholinesterases (AChEs) were extracted from two Mexican Boophilus microplus strains that demonstrated resistance to the organophosphate (OP) acaricide, coumaphos, in bioassay. The rate of inhibition of the extracted AChEs by the diethyl-OP paraoxon was determined for two resistant strains and two susceptible strains of B. microplus. The time to inhibition of 50% AChE activity was approximately two-fold greater for the resistant strains. Kinetic analysis of the interaction of the resistant AChEs with paraoxon revealed reduced bimolecular reaction constants (ki). Apparent conformational changes in the AChE of the resistant strains were reflected in reduced Km and Vmax values. The bimolecular reaction constants (ki) of the resistant strains were most affected by a slower rate of enzyme phosphorylation (k2).     

Identification of two acetylcholinesterases in Pardosa pseudoannulata and the sensitivity to insecticides

Pardosa pseudoannulata is an important predatory enemy against insect pests, such as rice planthoppers and leafhoppers. In order to understand the insecticide selectivity between P. pseudoannulata and insect pests, two acetylcholinesterase genes, Pp-ace1 and Pp-ace2, were cloned from this natural enemy. The putative proteins encoded by Pp-ace1 and Pp-ace2 showed high similarities to insect AChE1 (63% to Liposcelis entomophila AChE1) and AChE2 (36% to Culex quinquefasciatus AChE2) with specific functional motifs, which indicated that two genes might encode AChE1 and AChE2 proteins respectively. The recombinant proteins by expressing Pp-ace1 and Pp-ace2 genes in insect sf9 cells showed high AChE activities. The kinetic parameters, V_max and K_m, of two recombinant AChE proteins were significantly different. The sensitivities to six insecticides were determined in two recombinant AChEs. Pp-AChE1 was more sensitive to all tested insecticides than Pp-AChE2, such as fenobucarb (54 times in K_i ratios), isoprocarb (31 times), carbaryl (13 times) and omethoate (6 times). These results indicated that Pp-AChE1 might be the major synaptic enzyme in the spider. By sequence comparison of P. pseudoannulata and insect AChEs, the key amino acid differences at or close to the functional sites were found. The locations of some key amino acid differences were consistent with the point mutation sites in insect AChEs that were associated with insecticide resistance, such as Phe331 in Pp-AChE2 corresponding to Ser331Phe mutation in Myzus persicae and Aphis gossypii AChE2, which might play important roles in insecticide selectivity between P. pseudoannulata and insect pests. Of course, the direct evidences are needed through further studies.     

BIOCHEMICAL ASPECTS OF INSECTICIDE RESISTANCE IN COTTON BOLLWORM FROM HANDAN OF HEBEI PROVINCE*

Abstract The cotton bollworms, Helicoverpa armigera Hübner, collected from Handan of Hebei Province, have evolved high resistance to pyrethroid, organophosphate and carbamate insecticides, The sensitivity of acetylcholinesterase (AChEs) to paraoxon and methomyl varied with the development stages of the cotton bollworm. After the treatments with LD₅ and LD₅₀ of parathion and methomyl to the cotton bollworms, the affinity of AChE to acetylthiocholine (ATCh) and acetyl-β-bmethyl-thio choline (MeTCh) increased significantly except the treatment of parathion using LD₅₀ dosage while the sensitivity of AChEs to paraoxon significantly decreased. The sensitivity of AChEs to methomyl strongly increased in the treatment of parathion using LD₅₀ dosages while strongly decreased in other treatments. The affinity of carboxylesterase to β-naphthyl acetate (β-NA) was higher in groups of treatment with insecticides than in group of control. The glutathione-s-transferase (GST) activity significantly decreased in the induced groups using LD₅ dosages, while increased in the selection groups using LD₅₀ dosages. The effects of parathion and methomyl on the phosphatases of cotton bollworm were related to the dosages of application and the time after treatment and the effect on the alkaline phosphatase was stronger than on acid phosphatase.     

Comparative studies of acetylcholinesterase purified from three field populations of Liposcelis entomophila (enderlein) (psocoptera: liposcelididae)

Acetylcholinesterace (AChE) is known to be the major target for organophophate and carbamate insecticides and biomolecular changes to AChE have been demonstrated to be an important mechanism for insecticide resistance in many insect species. In this study, AChE from three field populations of Liposcelis entomophila (Enderlein) (Psocoptera: Liposcelididae) was purified by affinity chromatography and subsequently characterized by its Michaelis‐Menten kinetics to determine if detectable changes to AChE have occurred. Bioassays revealed that the potential resistance threat of psocids in Sichuan Province (GH) was greater than either Hubei Province (WH) or Chongqing Municipality (BB). Compared to the other two populations, the WH population possessed the highest specific activity of purified AChE. Kinetic analyses indicated that the purified AChE from GH population expressed a significantly lower affinity to the substrate and a higher catalytic activity toward acetylthiocholine iodide (ATChI) (i.e., higher K_m and V_max values) than BB and WH populations. In vitro studies of AChE suggest that five inhibitors (aldicarb, eserine, BW284C51, omethoate, and propoxur) all possess strong inhibitory effects with eserine having the strongest inhibitory effect against purified AChE. According to bimolecular rate constants (k_i), the purified AChE from GH population was least sensitive to all inhibitors except for omethoate. The differences in AChE among the three populations may be partially attributed to the differences in pesticide application and control practices for psocids among the three locations. © 2010 Wiley Periodicals, Inc.     

Expression of two types of acetylcholinesterase gene from the silkworm, Bombyx mori, in insect cells

Complementary DNAs encoding two types of acetylcholinesterase （ACHE） were isolated from the silkworm, Bombyx mori. The type 1 （Bmacel） and type 2 （Bmace2） ORFs are 2052 and 1917 bp in length, respectively. Both the complete ORFs of the Bmaces and C- terminal truncated forms were recombined into the Bacmid baculovirus vector under the control of the polyhedrin promoter and expressed in Trichoplusia ni （Tn-5B 1-4） cells. The resulting products exhibited ACHE activity and glycosylation of the expressed proteins. An inhibition assay indicated that the ace2-type enzyme was more sensitive than the acel-type enzyme to inhibition by eserine and paraoxon.     

Contribution to the genetics of serum β-lipoproteins in man

Summary The genetic behaviour of a human serum -lipoprotein factor, called Ag(a₁), was studied by agar micro-diffusion technique, utilizing an antibody detected in the serum from a transfused thalassemia patient. It behaves as an inherited, dominant, autosomal character, with complete penetrance at birth. It is controlled by a gene and is closely linked to the Ag ^x and Ag ^y genes.The existence of a gene Ag ^b , allelic to , is postulated but the Ag(b) antigen has not so far been detected by specific antisera.The frequency of the gene in a Milan population was found to be 0,43, and in a Berne population was 0,46. The frequencies of the four possible gene combinations in the sample group from Milan were: Ag ^yb =0,53; =0,22; =0,21; Ag ^xb =0,04.The observed frequencies of the factor Ag(a₁) were 0,676 and 0,713, respectively among the Milan and Berne populations.     

Purification and Characterization of Carboxylesterase from the Seeds of Jatropha curcas

Carboxylesterases are hydrolases which catalyze the hydrolysis of various types of esters. Carboxylesterase from the seeds of Jatropha curcas has been purified to homogeneity using ammonium sulfate fractionation, CM-cellulose chromatography, Sephadex G-100 chromatography and preparative polyacrylamide gel electrophoresis (PAGE). The homogeneity of the purified enzyme was confirmed by PAGE, iso-electrofocusing and SDS-PAGE. The molecular weight of the purified enzyme was determined by both gel-permeation chromatography on Sephadex G-150 and SDS-PAGE. The molecular weight determined by Sephadex G-150 chromatography and SDS-PAGE both in the presence and absence of 2-mercaptoethanol was 31 kDa. The isoelectric point of the purified enzyme was found to be 8.9. JCSE-I (J. curcas seed esterase-I) was classified as carboxylesterase on the basis of substrate and inhibitor specificity. The K_m of JCSE-I with 1-naphthyl acetate, 1-naphthyl propionate, 1-naphthyl butyrate and 2-naphthyl acetate as substrates were found to be 0.0,794, 0.0,658, 0.0,567 and 0.1 mM, respectively. The enzyme exhibited an optimum temperature of 45 °C and an optimum pH of 6.5. The enzyme was stable up to 15 min at 65 °C. The enzyme was resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB) and inhibited by organophosphates (dichlorvos, parathion and phosphamidon).     

STUDIES ON THE KINETICS OF ACETYLCHOLINESTERASE IN THE RESISIANT AND SUSCEPTIBLE STRAINS OF HOUSEFLY (MUSCA DOMESTICA)

Abstract Acetylcholinesterase (AChE) in the susceptible (S) and the resistant (R) strains of housefly (Musca domestica) was investigated using kinetic analysis. The V_max values of AChE for hydrolyzing acetylthiocholine (ATCh) and butyrylthiocholine (BTCh) were 4578.50 and 1716.08nmol/min/mg* protein in the R strain, and were 1884.75 and 864.72 nmol/min/mg. protein in the Sstrain, respectively. The V_max ratios of R to S enzyme were 2.43 for ATCh and 1.98 for BTCh. The K_m values of AChE for ATCh and BTCh were 0.069 and 0.034 mmol/L in the S strain, and 0.156, 0.059 mmol/L in the R strain, respectively. The K_m ratios of R to S enzyme were 2.26 for ATCh and 1.74 for BTCh. The k_i ratios of S to R enzyme for three insecticides propoxur, methomyl and paraoxon were 46.04, 4.17 and 2. 86, respectively. In addition, k_cat and k_cat/K_m for measuring turnover and catalytic efficiency of AChE were determined using eserine as titrant. The k_cat values of AChE from the R strain for both ATCh and BTCh were higher than those values from the S strain. But the values of k_cat/K_m were in contrary to the k_cat values with R enzyme compared to S enzyme. The AChE catalytic properties and sensitivity to the inhibition by three insecticides in the R and S strains of housefly were discussed based on contribution of V_max, K_m, k_i, k_cat and k_cat/K_m. All these data implied that AChE from the R strain might be qualitatively altered. We also observed an intriguing phenomenon that inhibitors could enhance the activity of AChE from the resistant strain. This “flight reaction” of the powerful enzyme might be correlated with the developing resistance of housefly to organophosphate or carbamate insecticides.     

Pharmacodynamics of malathion and carbaryl in susceptible and multiresistant German cockroaches (Dictyoptera: Blattellidae)

Studies with malathion and carbaryl were done to compare toxicity; absorption, metabolism, internal accumulation, and excretion; and in vivo inhibition of acetylcholinesterase (AChE) after topical applications to adult male susceptible (S, Orlando normal) or multiresistant (R, HRDC) German cockroaches, Blattella germanica (L.). Compared with the S strain, R cockroaches were highly resistant to malathion (about 33-fold) and only moderately resistant or tolerant to carbaryl (about 5-fold). Tests with topically applied 14C-labeled malathion and carbaryl indicated that both compounds penetrated rapidly and radioactive products were readily excreted. Rates of absorption or excretion in S and R strains did not differ significantly. Both insecticides were extensively metabolized; each yielded the same array and similar concentrations of metabolites in insects from either strain. In contrast, metabolic detoxification of malathion and carbaryl was significantly greater in R cockroaches when the insects were treated by injection. Strains did not differ significantly in the in vitro inhibition of brain AChE by either malaoxon or carbaryl. However, dramatic differences were observed between strains in the in vivo inhibition of AChE during a 6-h test period after topical treatment with malathion, and moderate but significant differences occurred between strains in the in vivo inhibition of AChE by carbaryl. These data suggest that the strong resistance to malathion and moderate resistance or tolerance to carbaryl in R cockroaches is probably a result of enhanced capability for metabolic detoxification.     

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.     

Toxicity assessment of insecticides commonly used in rice pest management to the fry of common carp, Cyprinus carpio, a food fish culturable in rice fields

Toxicity of four insecticides commonly used in rice pest management, chlorpyrifos, dimethoate, carbaryl and carbosulfan, to the fry of common carp was assessed through median lethal concentrations (LC₅₀) and in vivo inhibition of the brain acetylcholinesterase (AChE) enzyme at sublethal concentrations. The 96‐h LC₅₀ values for these four insecticides were determined to be 0.008, 26.11, 7.85 and 0.60 mg L^?1 respectively. Exposure of fish to a series of sublethal concentrations (0.5–5% LC₅₀) of each insecticide for 14 days resulted in concentration‐dependent inhibition in AChE activity in comparison with the controls. AChE activity was greatly inhibited in the fish exposed to sublethal concentrations of chlorpyrifos. Upon transfer to insecticide‐free water, AChE activities in fry exposed to 0.5 and 1% LC₅₀ concentrations of carbaryl and carbosulfan were restored to the control level within 7–21 days whereas the fish exposed to chlorpyrifos or dimethoate did not fully recover from the insecticide‐induced anticholinesterase action. Of the four insecticides tested, chlorpyrifos was the most toxic for the fry of common carp. Although dimethoate was least toxic for the fish under acute exposure, the restoration level of normal AChE activity was slower under chronic exposure in comparison with carbaryl and carbosulfan. Hence, the use of carbamates, especially carbaryl, to control insect pests of rice in rice‐cum‐carp culture systems is recommended when considering survival, restoration of the normal AChE activity and stamina of the cultured fish.     

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.     

Various forms of mouse lactoferrins: purification and characterization

This work was conducted to study the microheterogeneity of mouse lactoferrin (LF). Two forms, LF₁ and LF₂, could be purified from uterine luminal fluid by ion-exchange HPLC on a Protein PAK SP 5PW column. Another form, LF₃, was purified from the epididymis homogenate by affinity chromatography on a column of Protein A-Sepharose coupled with the purified LF₂ antibody that was prepared to give no crossreaction with serum albumin. Both LF₁ and LF₂ showed a M_r 74 000 band while LF₃ gave a M_r 70 000 band on reducing SDS–PAGE. All of them were reduced to a M_r 68 000 band after they had been digested with N-glycosidase F. The data from automated Edman degradation confirmed the completely identical 19 amino acid sequences in the N-terminal regions of these three LFs, except the lack of N-terminal Lys–Ala of LF₂/LF₃ in LF₁. LF in tissue homogenates was immunodetected by Western blot procedure using the purified LF₂ antibody. Different amounts of LF with a molecular mass of the 70 000 or 74 000 were distributed in the non-sexual organs such as kidney, spleen, lung, heart and liver and the sexual glands including epididymis, vagina, uterus, ovary and prostate. No LF was detected in stomach, intestine, testis and seminal vesicle.     

Characterization of trimeric acetylcholinesterase from a legume plant, <Emphasis Type="Italic">Macroptilium atropurpureum</Emphasis> Urb.

We recently identified plant acetylcholinesterases (E.C.3.1.1.7; AChEs) homologous to the AChE purified from a monocotyledon, maize, that are distinct from the animal AChE family. In this study, we purified, cloned and characterized an AChE from a dicotyledon, siratro. The full-length cDNA of siratro AChE is 1,441 nucleotides, encoding a 382-residue protein that includes a signal peptide. This AChE is a disulfide-linked 125-kDa homotrimer consisting of 41–42 kDa subunits, in contrast to the maize AChE, which exists as a mixture of disulfide and non-covalently linked 88-kDa homodimers. The plant AChEs apparently consist of various quaternary structures, depending on the plant species, similar to the animal AChEs. We compared the enzymatic properties of the dimeric maize and trimeric siratro AChEs. Similar to electric eel AChE, both plant AChEs hydrolyzed acetylthiocholine (or acetylcholine) and propionylthiocholine (or propionylcholine), but not butyrylthiocholine (or butyrylcholine), and their specificity constant was highest against acetylcholine. There was no significant difference between the enzymatic properties of trimeric and dimeric AChEs, although two plant AChEs had low substrate turnover numbers compared with electric eel AChE. The two plant AChE activities were not inhibited by excess substrate concentrations. Thus, similar to some plant AChEs, siratro and maize AChEs showed enzymatic properties of both animal AChE and animal BChE. On the other hand, both siratro and maize AChEs exhibited low sensitivity to the AChE-specific inhibitor neostigmine bromide, dissimilar to other plant AChEs. These differences in enzymatic properties of plant AChEs may reflect the phylogenetic evolution of AChEs. Kosuke Yamamoto and Yoshie S. Momonoki contributed equally to this work.