Binding Sites for Ca2+-Channel Effectors and Ryanodine in Periplaneta americana—Possible Targets for New Insecticides

The calcium channel and the ‘calcium release channel’ of muscle membrane of the cockroach Periplaneta americana have been characterized. Biological assays with calcium channel blockers and ryanodine on different insects and acari revealed pronounced insecticidal effects with ryanodine, but not with calcium channel blockers, at concentrations between 0·1 and 300 μg ml⁻¹. Skeletal muscle membranes derived either from the tubular network or from the sarcoplasmatic reticulum of P. americana were characterized with respect to the binding of the dihydropyridine (DHP) [³H]isradipine (PN 200-110), the phenyl-alkylamine [³H]verapamil and the alkaloid [³H]ryanodine. Preliminary binding studies with the benzothiazepine [³H]diltiazem suggest a low-affinity binding site with a IC₅₀ value of 3·3 μM . All binding sites tested were sensitive to treatment with proteinase K. Optimal conditions for binding of the radioligand ryanodine revealed the highest specific binding at pH 8 and at calcium chloride concentrations between 100 and 500 μM . EGTA at 10 μM abolished 95% of the ryanodine binding. Binding studies with calcium channel binding sites revealed a pronounced effect of low Ca²⁺ concentrations on specific isradipine binding, whereas verapamil and diltiazem binding were only reduced by the presence of 200 μM EGTA. With respect to high Ca²⁺ concentrations, specific binding of diltiazem, isradipine and verapamil was reduced by 73, 40 and 20%, respectively, at 5 mM Ca²⁺. Radioligand binding experiments showed high-affinity binding sites for ryanodine and isradipine. K_D values of 0·95 nM (B_max=550 fmol mg⁻¹ protein) and 0·75 nM (B_max=213 fmol mg⁻¹ protein) were determined respectively. A lower-affinity binding site was identified in binding studies with verapamil (K_D=7·4 nM and B_max=27 fmol mg⁻¹ protein). [³H]isradipine displacement studies with several dihydropyridines revealed the following ranking of affinity: nitrendipine>isradipine>Bay K8664≪nicardipine. Displacement of [³H]verapamil binding by effectors of the phenylalkylamine binding site showed that bepridil and S(-)verapamil had the highest affinities of the compounds tested followed by (±)verapamil, nor-methylverapamil and R(+)verapamil.     

Inhibitors of choline acetyltransferase as potential insecticides

Choline acetyltransferase (E.C.2.3.1.6) catalyses the synthesis of acetylcholine and is therefore a target for a new insecticide. We have prepared a variety of compounds, mainly choline analogues, as inhibitors of this enzyme. One of these, 2-isothiocyanatoethyltrimethylammonium iodide, has a K_i of 0.06 μM (K_m for choline is 150 μM ) and is apparently the most powerful inhibitor known for this enzyme. Although some of our compounds are insecticidal we believe, on the basis of electrophysiological studies, that they act, not on choline acetyltransferase, but on the acetylcholine receptor of the insect.     

Influence of Pesticides on UDP-glucuronosyltransferase in Rat Liver Microsomes

Fifteen pesticides, representatives of different chemical groups, were tested for their inhibitory effect on the glucuronidation of 4-nitrophenol (4-NP) and phenolphthalein (PPh) by rat liver microsomes. Three herbicides (simazine, chlorsulfuron, tribenuron-methyl), two insecticides (dioxacarb, carbaryl) and one fungicide (zineb) significantly decreased the UDP-glucuronosyltransferase (UDPGT) activity. The carbamate insecticide dioxacarb was found to be the most potent inhibitor, at 1 mM concentration suppressing 4-NP-UDPGT activity completely, and reducing by 55% the activity associated with the conjugation of PPh. One millimole simazine and carbaryl affected only 4-NP glucuronidation, while chlorsulfuron and zineb exerted a marked inhibition of both 4-NP and PPh conversion. Concentrations of 0·1 mM carbaryl, dioxacarb and zineb were still inhibitory against 4-NP-UDPGT, with zineb producing 40% inhibition of PPh glucuronidation. As a whole, UDPGT isoforms conjugating PPh were less sensitive to the agrochemicals tested. Kinetic studies with dioxacarb, chlorsulfuron and carbaryl revealed a mixed type of inhibition with respect to the acceptor substrate 4-NP, with apparentK_i values of 70 μM , 120 μM and 160 μM , respectively.     

Firefly luciferase inhibition by isopropyl-3-chlorocarbanilate and isopropyl-3-chloro-hydroxycarbanilate analogues

The firefly luciferase ATP assay was inhibited by the herbicide, isopropyl-3-chlorocarbanilate (I), and by two of its hydroxylated metabolites, isopropyl-5-chloro-2-hydroxycarbanilate (II) and isopropyl-3-chloro-4-hydroxycarbanilate (III). The β-O-glucosides of II and III reversed the inhibition of luciferase. Compounds I and II were linear noncompetitive inhibitors in respect to ATP (K_i ? 20 μM, each) and were linear competive inhibitors in respect to d-luciferin (K_i ? 6 μM, each). Compound III was a linear competitive inhibitor in respect to both ATP and d-luciferin (K_i ? 1 and 6 μM, respectively). The inhibition caused by III appeared to remain competitive for both substrates when AMP was added to the system, but the inhibition exhibited by III with respect to ATP and d-luciferin was more effective (K_i ? 0.5 μM, each). The effects of compounds I, II, and III upon the firefly luciferase ATP assay are discussed, and a relationship between the firefly system and plant susceptibility to compound I is proposed.     

Quantitative autoradiography of GABA receptors in locust (Schistocerca americana) brain

Conventional film autoradiography was used at the light microscopic level for the localization and quantization of 4-aminobutyric acid (GABA) receptors in the locust brain (Schistocerca americana). Localization of the receptor site was achieved via binding with the receptor-ligand probe [³ H]muscimol. Frozen sections were cut and subsequently incubated either in 40 nM [³H]muscimol or by coincubating sections with [³H]muscimol and one of the following: GABA (50 μM)], a receptor specific agonist [muscimol (1 μM) or isoguvacine (1 μM)], an uptake inhibitor [nipecotic acid (50 μM)], or a noncompetitive channel modulator [avermectin B_1a, (1 μM) or aldrin (50 μM)]. Through computer image enhancement and densitometric analysis of the optical density of [³H]muscimol binding sites, the interaction of the above compounds with the putative GABA receptor was determined for various anatomical regions of the locust brain. By comparing the differently treated, but adjacent sections, GABA receptor distribution was quantitated and mapped. Receptor sites were found distributed in the antennal lobes, central body, β-lobe and β-lobe of the corpus pedunculatum, protocerebral bridge, and calyx as well as the optic lobe regions.     

Effect of miconazole,an antifungal agent,on allene oxide synthase: Inhibition,kinetics, and binding

We investigated the inhibition of allene oxide synthase (AOS), a key enzyme in jasmonic acid biosynthesis, by miconazole. Kinetic analysis indicated that miconazole was a mixed-type inhibitor of AOS with a K_i value of approximately 8.4 ± 0.2 μM. Analysis of the interactions between miconazole and AOS by optical difference spectroscopy revealed that miconazole binding induces type II binding spectra with a K_d value of approximately 6.0 ± 0.2 μM.     

Phosphinate inhibition studies of cholinesterases

The kinetic constants, K_d, k₂, and k_i, were determined for the inhibition by 4-nitro-phenyl methyl(phenyl)phosphinate of three cholinesterases: butyrylcholinesterase, bovine erythrocyte acetylcholinesterase and eel acetylcholinesterase. Stopped-flow kinetic evaluations and automated data acquisition and processing were employed. A broad range in affinity for the phosphinate inhibitor was observed as reflected by the binding constants, K_d. A similar wide range in the k₂ values for the unimolecular inhibition step was obtained. The net bimolecular rate constants, ki, indicate equal overall reactivity for butyrylcholinesterase and eel acetylcholinesterase with a smaller inhibition rate constant for bovine erythrocyte acetylcholinesterase.     

[3H]Muscimol binding to a putative GABA receptor in honey bee brain and its interaction with avermectin B1a

A putative GABA receptor was identified in honey bee brain by virtue of its specific binding of [³H]muscimol and its drug specificity. [³H]Muscimol bound with two affinities (K_d1 of 3 nM and K_d2 of 144 nM), comparable to its affinities for binding to mammalian brain. The high-affinity binding was most sensitive to GABA agonists with the following decreasing order of potencies: muscimol>GABA>imidazole acetic acid>DL-GABOB>Zβ-guanidine propionic acid. However, it was insensitive to the antagonist bicuculline, which is potent on [³H]muscimol binding to the mammalian GABA_A receptor. It was also insensitive to baclofen, which is a potent agonist of mammalian GABA_B receptor, as well as to picrotoxinin, pentobarbital, flunitrazepam, and ethyl-β-carboxylate, which bind to allosteric sites in mammalian GABA receptor. The low-affinity [³H]muscimol binding was inhibited with GABA agonists with the following decreasing order of potencies: imidazole acetic acid = β-guanidine propionic acid>dl-GABOB. The two muscimol binding affinities may represent binding to two sites on the same GABA receptor or to two kinds of GABA receptor. The most potent inhibitor of the high-affinity [³H]muscimol binding to honey bee brain was avermectin B_1a (AVM), whose IC₅₀ was 0.01 nM. AVM also inhibited the low-affinity [³H]muscimol binding with an IC₅₀ of 2 μM.     

Pesticide transformation by a variant of CYPBM3 with improved peroxygenase activity

The activity of the mutant CYPBM3 “21B3”, which is able to use hydrogen peroxide as the final electron acceptor, was evaluated against two major environmental pollutants; organochlorine and organophosphorus pesticides. This evolved CYP from Bacillus megaterium is able to transform a variety of structurally different pesticides. The catalytic parameters for two organochlorine; dichlorophen (k_cat = 9.2 min⁻¹, K_M = 64.1 μM) and linuron (k_cat = 226.5 min⁻¹, K_M = 468.2 μM), and two organophosphorus compounds; parathion (k_cat = 10.9 min⁻¹, K_M = 59.3 μM) and chlorpyrifos (k_cat = 9.2 min⁻¹, K_M = 226.5 μM) were determined giving catalytic efficiencies between 0.143 and 1.107 min⁻¹ μM⁻¹. CYPBM3 “21B3” has the ability to both activate and detoxify organophosphorus pesticides, as demonstrated by the chemical nature of the reaction products. The capacity to transform structurally diverse compounds together with the great stability, easy production and relatively inexpensive cofactors needed, makes CYPBM3 “21B3” an enzyme with a potential use on the environmental field.     

Substrate binding by blasticidin S deaminase,an aminohydrolase for novel 4-aminopyrimidine nucleosides

The substrate specificity of blasticidin S deaminase (E.C. 3.5.4.23), purified from Aspergillus terreus, has been studied in detail. The enzyme was found to catalyze the hydrolytic deamination of cytosine nucleus in blasticidin S and its derivatives, while cytosine, cytidine, cytidine monophosphate, adenosine, and guanosine were not regarded as substrates. The structural requirements of compounds for binding by the enzyme were evaluated by the nature of inhibition and inhibitor constants; deaminohydroxyblasticidin S, the reaction product, and its derivatives inhibited the enzymatic aminohydrolysis of blasticidin S competitively. The K_i for deaminohydroxyblasticidin S was determined to be 2.3 × 10⁻⁵ M, which is close to the K_m for blasticidin S (2.1 × 10⁻⁵ M). The binding affinity of derivatives to the enzyme, −ΔG_bind, was calculated on the basis of K_i values, and further the partial binding affinities, −Δg, for several moieties or atomic groups in blasticidin S were deduced from the −ΔG_bind values. The results showed that the enzyme involves a specific binding site with multiple points corresponding to the carboxyl group of cytosinine moiety, the amide group, and the β-amino and guanidino groups of blastidic acid moiety in blasticidin S molecule; these facts are strongly indicative of the enzyme to be a new aminohydrolase for novel nucleosides such as blasticidin S.     

Inhibitory effect of valienamine on the enzymatic activity of honeybee (Apis cerana Fabr.) α-glucosidase

Valienamine, an aminocyclitol with similar configuration to α-glucose, has a strong inhibitory effect on α-glucosidase. α-Glucosidase plays an important role in insect carbohydrate metabolism. The inhibitory effect of valienamine on the enzymatic activity of honeybee (Apis cerana Fabr.) α-glucosidase was investigated. Our results show that valienamine inhibition of honeybee α-glucosidase was pH- and dose-dependent, but temperature-independent. Valienamine is shown to be a potent and competitive reversible inhibitor of honeybee α-glucosidase in vitro with an IC₅₀ value of 5.22 × 10⁻⁵ M and K_i value of 3.54 × 10⁻⁴ M at pH 6.5, 45 °C. Valienamine has the potential to be developed into novel insecticides.     

Presence of Muscarinic Acetylcholine Receptors in the Cattle Tick Boophilus microplus and in Epithelial Tissue Culture Cells of Chironomus tentans

A muscarinic acetylcholine receptor (mAChR) has been demonstrated and partially characterized in larvae of the cattle tick Boophilus microplus. Its properties are compared with mAChR from an epithelial cell line from the dipteran insect Chironomus tentans. Competition studies with cholinergic ligands of different specificity revealed the muscarinic nature of the cholinergic receptors investigated in both species. In homogenates from tick larvae, specific binding sites for [³H]quinuclidinyl benzilate (QNB) with high affinity (1·2±(0·13) nM ; B_max 22·5 pmol mg protein⁻¹) were detected that do not bind nicotinic compounds specifically. The estimated IC₅₀ values for nicotine, imidacloprid and α-bungarotoxin were all in the mM range. Additionally, with tick larvae, high-affinity nicotinic binding sites were detected with [³H]nicotine which could be displaced by high concentrations of imidacloprid or QNB. The estimated IC₅₀ values for nicotine, α-bungarotoxin, imidacloprid and QNB were 43(±8) nM , 0·8(±0·2) μM , 2·8(±0·6) μM and 78(±1·9) μM , respectively. With homogenates of the non-neuronal insect cell line from C. tentans, only high-affinity binding sites for [³H]QNB were found. Muscarinic antagonists selectively displaced [³H]quinuclidinyl benzilate (QNB) binding to tick larvae homogenates. The mAChR of B. microplus preferred pirenzepine (IC₅₀ 2·13(±1·02) μM ) among different subtype-specific mAChR antagonists (4-DAMP had IC₅₀ 49·9(±9·13) μM and methoctramine had IC₅₀ 121(±14·2) μM ) indicating a type of binding site similar to the vertebrate M1 mAChR subtype. The tick muscarinic receptor seems to be a G-protein-coupled receptor, as concluded from the 4·8-fold reduction in receptor affinity for binding of the muscarinic agonist oxotremorine M upon treatment with the non-hydrolysable GTP-analogue γ-S-GTP. Binding data for the agonists oxotremorine M (IC₅₀ 71·3(±19·6) μM ) and carbachol (IC₅₀ 253(±87·1) μM ) parallel the biological efficacy of these compounds, in that, while oxotremorine M showed some activity against ticks, carbachol was ineffective.     

Sorption of lonisable organic compounds by field soils. Part 2: Cations,bases and zwitterions

Sorption of a range of permanent cations, bases and zwitterionic compounds was measured as a function of pH in two soil types. Pyridinium cations were more strongly sorbed (K_d 10– > 1000) than aliphatic cations (K_d < 5). At very low pH, sorption of the aliphatic cations sharply decreased, probably because they are displaced by protons. Most weak bases, including carbendazim andpyridines, were strongly sorbed (K_d 9–35) at low pH, where they would be appreciably protonated, sorption becoming much weaker at soil pH values > 6. However, an additional mechanism of sorption was observed for those zwitterions capable of chelation (e.g. picolinic acid and alanine) which gave rise to high K_d values at pH values near neutral. Inorganic phosphate was strongly sorbed (K_d > 140) save at very low pff. Glyphosate and inorganic phosphate were sorbed very strongly at pH values near to 4 (K_d > 200). The very strong sorption was attributed to ligand exchange interaction. Sorption of picolinic acid was similar when measured in water or calcium chloride solution (0.01 M). However, sorption decreased with increasing concentration of calcium chloride up to 1 M, probably because the protonated form of picolinic acid was displaced by calcium ions.     

Binding characteristics of methyl parathion to photosynthetic membranes of Chlorella

When methyl parathion is added at a given concentration to exponentially growing Chlorella cultures containing different numbers of cells, the inhibition of cell number, packed cell volume, pigment content, or photosynthesis has been found to be a function of the cell number, the inhibition being decreased with increased number of cells. Inhibition of photosynthesis has been studied further with a view to characterizing the mechanism of inhibition with a simple assumption that methyl parathion binds to a specific component in Chlorella cells to form an inhibitory complex. The concentration of methyl parathion causing 50% inhibition (I₅₀) of photosynthetic O₂ evolution increases linearly with increasing concentration of chlorophyll in the culture medium. With whole cells the inhibition constant (K_i) is 15 times greater than that with cell-free photosynthetic membranes. This shows that the cell wall acts as a permeability barrier. The relation between the I₅₀ and K_i values and the analyses of the Hill plot of the inhibition curves reveal one binding site per 0.5 chlorophyll molecule and a cooperative binding of methyl parathion with at least three binding sites per binding molecule. Mild treatment of the photosynthetic membranes with trypsin makes the photosynthetic electron transport insensitive to the insecticide, suggesting that the binding component is proteinaceous in nature and the binding sites are located on the external surface of the membrane. The reversal of methyl parathion inhibition is parallel to that of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (diuron) inhibition during trypsin treatment suggesting that the binding proteins for these two inhibitors are similar.     

Kinetic constants for the inhibition of acetylcholinesterase by phenyl carbamates

The kinetic constants of a variety of substituted phenyl N-methyl- and N,N-dimethylcarbamates, which inhibit bovine erythrocyte acetylcholinesterase, were determined by various experimental procedures. A procedure in the presence of a chromogenic substrate was developed, based on the suggestion of Hart and O'Brien, and was compared with the conventional Main method. The dissociation equilibrium constant, K_d, and the carbamylation rate constant, k₂, were shown to apparently depend on the inhibitor concentration range used for the determination in both procedures. Assuming that the binding of further inhibitor molecules to the reversible complex and the carbamylated enzyme is significant under conditions with high inhibitor concentrations, the concentration dependence of the kinetic constants was nicely delineated. It is indicated that reliable constants are obtainable with a rather low inhibitor concentration range, whose product by k_i is of the order of 0.2–1.0 min^?1.     

Microbial metabolism of propanil and 3,4-dichloroaniline

A Pseudomonas sp. which grew on 4-chloroaniline as a sole source of carbon and nitrogen was able to degrade 15% of 0.05 mM [¹⁴C]3,4-dichloroaniline to ¹⁴CO₂ within 10 days in presence of 1.5 mM 4-chloroaniline. The catabolic enzymes which degraded 3,4-dichloroaniline to CO₂ were inducible by 4-chloroaniline and by 3,4-dichloroaniline. However, their activity was much lower on 3,4-dichloroaniline than on 4-chloroaniline. The strain showed no significant growth on 3,4-dichloroaniline as a sole source of carbon and nitrogen. Soils supplemented with [ring-¹⁴C]propanil and the Pseudomonas sp. evolved 25–50% ¹⁴CO₂ within 5 days. The ¹⁴CO₂ evolution remained below 1% in absence of the Pseudomonas sp.     

Synthesis and thylakoid membrane binding of the radioactively labeled herbicide dinoseb

2,4-Dinitro-6-isobutylphenol (i-dinoseb), an isomer of the phenolic herbicide dinoseb and equally active as an inhibitor of photosynthetic electron transport and photophosphorylation, has been synthesized, ³H-labeled with a specific activity of 490 mCi/mmol. Its binding to broken chloroplasts is strongly pH dependent and biphasic representing a high- and a low-affinity binding site. For specific binding of i-dinoseb a binding constant K_b = 6.9 × 10^?8M has been determined. The number of binding sites corresponds to one molecule i-dinoseb per 830 molecules of chlorophyll, i.e., one molecule per two electron transport chains. i-Dinoseb can be displaced from the thylakoid membrane by DCMU-type inhibitors, and inhibitory uncouplers, but not by DBMIB-type inhibitors and uncouplers of oxidative phosphorylation. An extensive analysis of displacement by DCMU-type and phenolic herbicides indicates that DCMU-type herbicides interfere noncompetetively but phenolic herbicides interfere competetively with the specific binding of i-Dinoseb. It is concluded, therefore, that the binding sites of both types of herbicides are not identical although they are located on the same protein. The specific binding constant of i-dinoseb does not change in trypsin-treated chloroplasts whereas the number of binding sites is slightly reduced.     

Effect of s-triazine and phenoxyalkanoic acid herbicides on UDP-glucuronosyltransferase in rat liver microsomes

Twelve herbicides, representatives of two chemical groups, substituted phenoxyalkanoic acids and s-triazines, were tested for their inhibitory effect on the glucuronidation of 4-nitrophenol (4-NP), phenolphthalein (PPh) and 4-methylumbelliferone (4-MU) by rat liver microsomes. One millimole MCPA, ametryn and cyanazine significantly decreased PPh UDP-glucuronosyltransferase (UGT) activity, while propazine was found to be a most potent inhibitor of 4-NP glucuronidation. Concentrations of 0.1 mM dichlorprop and cyanazine were still inhibitory against PPh-UGT. The inhibition of 4-MU glucuronidation by the herbicides was low and not specific. As a whole, s-triazine derivatives were more inhibitory than the substituted phenoxyalkanoic acids. Kinetic studies with propazine revealed a non-competitive type of inhibition towards the acceptor substrate 4-NP, with an apparent K_i value of 0.540 mM . With ametryn, an uncompetitive type of inhibition against PPh and a mixed type of inhibition towards UDPGA were found, with apparent K_i values of 0.330 mM and 0.380 mM , respectively. © 1999 Society of Chemical Industry     

Structure-activity relationships of pheromonostasis induced by ACCase-inhibitor herbicides in the moth Helicoverpa armigera

Many moth sex pheromone blends are derived from fatty acids and their production is regulated by a Pheromone Biosynthesis Activating Neuropeptide (PBAN). In prior work we showed that the herbicide Diclofop-acid, an acetyl-coenzyme A carboxylase (ACCase) inhibitor, inhibits PBAN-induced sex pheromone production in vitro. In this work we extend our study showing that several other herbicides, belonging to the 2-aryloxyphenoxypropionate (‘FOP’) and the cyclohexandione-oxime (‘DIM’) families significantly inhibit pheromone production by adult females whilst survival is unaffected by treatment. Enzyme activity in vitro and kinetic analysis revealed a K_m of 0.35 μM with K_i values of 0.1 and 0.28 μM due to Tralkoxydim and Diclofop inhibition, respectively. Inhibitory activity on PBAN-induced pheromone production by all herbicides tested revealed a potency order: Tralkoxydim > Clodinafop > Cycloxidim > Haloxyfop > Diclofop > Fenoxaprop > Fluazifop > Quizalofop, Quizalofop being inactive. Differences in inhibition efficiencies may be attributed to different binding sites on the enzyme or to the polarity and solubility of these compounds.     

Oxidation of chlorpyrifos, azinphos-methyl and phorate by myeloperoxidase

The present paper deals with the investigations of optimal conditions for the myeloperoxidase (MPO) mediated oxidation of chlorpyrifos, azinphos-methyl and phorate, organophosphorous pesticides (OPs) containing phosphorothionate group, from thio- to oxo-forms, in the presence of hydrogen peroxide. The aim of the work was to apply this oxidation method in the AChE based bioanalytical tests for OPs determination. The maximum concentration of oxo-forms for all tested pesticides was achieved after 10 min incubation of OPs in 50 mM phosphate buffer (pH 6.0) with 100 nM MPO in the presence of 50 μM H₂O₂. Optimal temperature for obtaining maximal concentration of oxo-forms was 37 °C. Only the parent compounds and their oxo-forms were identified chromatographically in the OPs samples after their exposure to MPO. Moreover, no hydrolysis products were detected in the time interval of 1 h after the MPO catalyzed reaction was stopped by catalase. The efficiency of OPs transformation from thio- to oxo-forms was measured using acethylcholinesterase (AChE) test, by comparison of percent of AChE inhibition before and after exposure to the oxidized sample.