Acetylcholinesterase from desert Cobra (Walterinnesia aegyptia) venom: Optimization and kinetics study

Acetylcholinesterase (AChE) was investigated inWalterinnesia aegyptia venom and characterized with respect to its kinetic properties. It was found that 4.0 ug of crude venom protein and an incubation time of 4.0 min were suitable conditions for linearity of AChE activity at 25°C. The optimum strength of the sodium phosphate buffer was 0.05 M, and the optimum pH was 7.75. The optimum temperature was 30°C. The activation energy and the heat of activation were observed to be 6510 and 5922 cal/mole. The AChE was specific for acetylthiocholine but it did not hydrolyse butyrylthiocholine. The optimum substrate concentration was 3.0 mM but at higher substrate concentrations, the AChE activity declined. The ASCh concentration ranges for different orders of the reactions were determined and kinetic parameters (K_m, V_max, k_cat, and k_sp) were established at each order of the reaction.Abbreviations AChE acetylcholinesterase - ASCh acetylthiocholine - K_m Michaelis-Menten constant - V_max the limiting maximal velocity - AChE_a acylated enzyme - k_cat turnover number - k_sp specificity constant     

Sensitivity of bovine retinal acetylcholinesterase (E.C. 3.1.1.7) toward tacrine: Kinetic characterization

This work addresses the kinetic analysis of the interaction of tacrine with bovine retina acetylcholinesterase (AChE, E.C. 3.1.1.7). It was found that the tacrine effect was reversible in nature. Tacrine inhibited bovine retinal AChE activity in a concentration-dependent manner; IC₅₀ was found to be 8.07 nM. The Michaelis-Menten constant (K_a) for the hydrolysis of acetylthiocholine iodide (ASCh) by AChE was 0.061 mM in the control system, and this value was increased by 54–67% in the tacrine-treated systems. The V_max was 0.701 μ mole/min per milligram protein for the control system, but it was decreased by 26–69% in the tacrine-treated systems. The Lineweaver–Burk plot, Dixon plot, and their secondary replots indicated that the nature of the inhibition was of the partial mixed type, that is, a mixture of competitive and noncompetitive inhibition. The values of K_i and K_t were estimated to be as 4.475 and 8.517 nM, respectively. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 245–251, 1998     

Kinetics of the inhibition of acetylcholinesterase from pigeon brain by procaine hydrochloride

The kinetic parameters of the inhibition of pigeon brain acetylchlolinesterase (AChE) by procaine hydrochloride were investigated. Procaine (0·083–1·67 mM) reversibly inhibited AChE activity (15–83 percent) in a concentration dependent manner, the IC₅₀ being about 0·38 mM. The Michaelis-Menten constant (K_m) for the hydrolysis of acetylthiocholine iodide was found to be 1·53 × 10^?4 M and the V_max was 1·06 μmol min^?1 mg^?1 protein. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition is of the linear mixed type which is considered to be a mixture of partial competitive and pure non-competitive. The values of K_i(slope) and K_i (intercepts) were estimated as 0·14 mM and 0·22 mM respectively by the primary Dixon and by the secondary replots of the Lineweaver-Burk plot. The K_i′/K_i ratio shows that procaine has a greater affinity of binding for the peripheral than for the active site.     

Comparative study of the inhibitory effect of antidepressants on cholinesterase activity in Bungarus sindanus (krait) venom,human serum and rat striatum

Cholinesterases are divided into two classes based on differences in their substrate specificity and tissue distribution: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). These enzymes may be inhibited by several compounds, such as antidepressants. The antidepressants paroxetine, imipramine, clomipramine and sertraline inhibited both venom AChE as well as human serum BChE in a concentration-dependent manner but had no effect on AChE in the rat brain striatum. The IC₅₀ of venom calculated for imipramine was 0.3 mM, paroxetine 0.38 mM, clomipramine 0.34 mM and sertraline 0.35 mM. Analysis of kinetic data indicated that the inhibition caused by sertraline and paroxetine was mixed, i.e. K_m values increased and V_max decreased in a concentration dependent manner. Imipramine and clomipramine exhibited competitive inhibition, i.e. K_m values increased and V_max remained constant. The present results suggest that these therapeutic agents used for depression can also be considered as inhibitors of snake venom and human serum cholinesterase.     

In vitro and in silico evaluation of fucosterol from Sargassum horridum as potential human acetylcholinesterase inhibitor

The fucosterol has been reported numerous biological activities. In this study, the activity in vitro of the fucosterol from Sargassum horridum as potential human acetylcholinesterase inhibitor was evaluated. The structural identification was obtained by nuclear magnetic resonance (NMR) spectroscopy and based on experimental data, we combined docking and molecular dynamics simulations coupled to the molecular-mechanics-generalized-born-surface-area approach to evaluating the structural and energetic basis for the molecular recognition of fucosterol and neostigmine at the binding site of acetylcholinesterase (AChE). In addition, the Lineweaver–Burk plot showed the nature of a non-competitive inhibition. The maximum velocity (V_max) and the constant of Michaelis–Menten (K_m) estimated for fucosterol (0.006 µM) were 0.015 1/V_o (ΔA/h and 6.399 1/[ACh] mM^?1, respectively. While, for neostigmine (0.14 µM), the V_max was 0.022 1/V_o (ΔA/h) and K_m of 6.726 1/[ACh] mM^?1, these results showed a more effective inhibition by fucosterol respect to neostigmine. Structural analysis revealed that neostigmine reaches the AChE binding site reported elsewhere, whereas fucosterol can act as a no-competitive and competitive acetylcholinesterase inhibitor, in agree with kinetic enzymatic experiments. Binding free energy calculations revealed that fucosterol reaches the acetylcholinesterase binding site with higher affinity than neostigmine, which is according to experimental results. Whereas the per-residue decomposition free energy analysis let us identify crucial residues involved in the molecular recognition of ligands by AChE. Results corroborate the ability of theoretical methods to provide crucial information at the atomic level about energetic and structural differences in the binding interaction and affinity from fucosterol with AChE.

Communicated by Ramaswamy H. Sarma     

On the inhibition of camel retina acetylcholinesterase activity by cycloheximide in vitro

The kinetic parameters of inhibition of camel retinal acetylcholinesterase (AChE) activity by cycloheximide (CH) were investigated. For the control system, the Michaelis–Menten constant (K _m)for the hydrolysis of acetylthiocholine iodide was found to be 0.076 mmol/L and the V _max was 0.547 mol/min per mg protein. In contrast, these parameters were decreased in the CH-treated systems. Dixon and Lineweaver–Burk plots, and their secondary replots, indicated that the inhibition was of the linear mixed type, which seems to be a combination of partial competitive and pure noncompetitive inhibition. The values of K_i(slope) and K _I(intercept) were estimated to be 3.50 and 5.68 mmol/L, respectively. K _i was greater than K_i, indicating that CH has a greater binding affinity for the peripheral site than the active site.     

Evaluation of 2,6-dichlorophenolindophenol acetate as a substrate for acetylcholinesterase activity assay

Abstract

Ellman’s method is a standard protocol for the determination of cholinesterases activity. Though the method is ready for laboratory purposes, it has some drawbacks as well. In the current article, 2,6-dichloroindophenol acetate is performed as a chromogenic substrate suitable for acetylcholinesterase (AChE) activity examination. Michaelis constant and maximal velocity for 2,6-dichloroindophenol acetate were determined (38.0?µM and 244 pkat) and compared to the values for acetythiocholine (K_m 0.18?mM; V_max 5.1?nkat). Docking for 2,6-dichloroindophenol acetate and human AChE was done as well. In conclusion, 2,6-dichloroindophenol acetate seems to be suitable chromogenic substrate for AChE and spectrophotometry and based on this it can be easily performed whenever AChE activity should be tested.     

Action of barbiturates on activity of acetylcholinesterase from synaptosomal membranes

The acetylcholinesterase from synaptosomal membranes is inhibited by anesthetics: Nembutal, brietal, and thiopental. Nembutal and brietal decrease theK _m for acetylthiocholine, without changes inV _max. A noncompetitive type of inhibition is produced by thiopental. This anesthetic decreases Arrhenius plot discontinuity by about 4°C and increases activation energies. Nembutal and brietal do not change Arrhenius plot discontinuities, but they increase activation energies. These results suggest that barbiturates change lipid-protein interactions in synaptosomal membranes.     

Catalytic properties of <Emphasis Type="Italic">Rhodotorula aurantiaca</Emphasis> KM-1 phenylalanine ammonia-lyase

L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) of the Rhodotorula aurantiaca strain KM-1 deaminates L-phenylalanine according to the Michaelis-Menten kinetics with K _M 1.75 ± 0.44 mM and V _max 3.01 ± 0.43 units/mg. The enzyme is competitively inhibited by D-phenylalanine with K _i 3.38 ± 0.32 mM. The Michaelis-Menten kinetics was analyzed, the inhibition type (competitive, noncompetitive, and mixed) was identified, and corresponding kinetic parameters were calculated using the computer programs written in Gauss 4.0. PAL was most stable at pH 6.55 and lacked approximately 50% of its activity after incubation at 57°C for 15 min. The yield of L-phenylalanine increased in the presence of mercaptoethanol, sodium ethylenediaminetetraacetate (EDTA), and ascorbic acid. The effects of EDTA and ascorbic acid were additive.     

Inactivation of acetylcholinesterase by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride

The neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to reversibly inhibit the activity of acetylcholinesterase. The inactivation of the enzyme was detected by monitoring the accumulation of yellow color produced from the reaction between thiocholine and dithiobisnitrobenzoate ion. The kinetic parameter, K _m for the substrate (acetylthiocholine), was found to be 0.216 mM and K _i for MPTP inactivation of acetylcholinesterase was found to be 2.14 mM. The inactivation of enzyme by MPTP was found to be dose-dependent. It was found that MPTP is neither a substrate of AChE nor the time-dependent inactivator. The studies of reaction kinetics indicate the inactivation of AChE to be a linear mixed-type inhibition. The dilution assays indicate that MPTP is a reversible inhibitor for AChE. These data suggest that once MPTP enters the basal ganglia of the brain, it can inactivate the acetylcholinesterase enzyme and thereby increase the acetylcholine level in the basal ganglia of brain, leading to potential cell dysfunction. It appears that the nigrostriatal toxicity by MPTP leading to Parkinson's disease-like syndrome may, in part, be mediated via the acetylcholinesterase inactivation.     

Acetylcholinesterase Activity and Enzyme Kinetics in the Brain of Common Carp,Cyprinus carpio Subjected to Sub-lethal Exposure to Dimethoate

Dimethoate, an organophosphorus insecticide, inhibited the acetylcholinesterase of the brain of a common carp, Cyprinus carpio, by increasing the K_m without affecting the V_max. A Dixon’s plot confirmed the competitive nature of the inhibition, yielding a K_i of 2 × 10^{? 3} M. The assay of brain acetylcholinesterase is thus useful in assessing pesticide toxicity to fishes.     

Effect of pyruvate on rat heart thiol status during ischemia and hypoxia followed by reperfusion

The effect of the neurotoxicant, 1-methyl-4-phenylpyridinium ion (MPP⁺) on acetylcholinesterase (AchE) activity was investigated. The MPP⁺ was found to inactivate the enzyme in a dose dependent manner. The kinetic parameter, K_m for the substrate (acetylthiocholine), was found to be 0.216 mM and K_i for MPP⁺ for the inactivation of AChE was found to be 0.197 mM. It was found that MPP⁺ is neither a substrate of AChE nor the time-dependent inactivator. The studies of reaction kinetics indicate inactivation of AChE to be a linear mixed-type inhibition. The inactivation of AChE by MPP⁺ was partially recovered by either dilution or gel exclusion chromatography. These data suggest that once MPP⁺ enters the basal ganglia of the brain, it can inactivate the AChE and thereby increase the acetylcholine level in the basal ganglia, leading to potential cell dysfunction. It appears likely that the nigrostriatal toxicity by MPP⁺ leading to Parkinson's disease-like syndrome may, in part, be mediated via the AChE inactivation.     

Kinetics of L-valine uptake in tobacco leaf discs. Comparison of wild-type,the digenic mutant Valr-2, and its monogenic derivatives

Uptake rates of L-valine in epidermis-free leaf discs of tobacco (Nicotiana tabacum L. cv. Xanthi) were measured over the concentration range 0.1 M to 50 mM. Wild-type tobacco was compared with the digenic mutant Val^r-2 (genotype vr2/vr2; vr3/vr3), and with the monogenic mutant strains h9 and h10 (genotype +/+; vr3/vr3) and h17 and h23 (genotype vr2/vr2; +/+). Rate equations consisting of one to three Michaelis-Menten terms, possibly in combination with a linear term were fitted to the kinetic data. These rate equations are equivalent to rational polynomials which may be regarded as the general type of mathematical function describing the kinetics of enzymes and carriers. Kinetic data of the four genotypes conformed to the sum of three Michaelis-Menten terms. Accordingly, three kinetic components could be distinguished. In the wild-type the approximate K_ms were 40 M, 1mM, and 40 mM, respectively. In Val^r-2 a component with a very low K_m (about 4 M) was found which may represent either the modified low-K_m component of the wild-type or a fourth component which is undetectable in the wild-type by kinetic analysis. The V_max of the low-K_m component in Val^r-2 was at least a 100-fold lower than in the wild-type. In the presence of one of the mutant genes the calculated V_max of the low-K_m component was 48% (strains h9 and h10) or 40% (strains h17 and h23) of the corresponding V_max in the wild-type. It is reasoned that the mutations have no effect on the activity of the other two kinetic components, though the evidence for this is circumstantial. Autoradiographs of leaf discs showed that in Val^r-2 the uptake of ¹⁴C-labelled valine in both mesophyll and minor veins was strongly reduced as compared with the wild-type.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DW dry weight - TPP⁺ tetraphenylphosphonium ion A preliminary account of part of this work has been presented (Borstlap 1986)     

The association between malathion resistance and acetylcholinesterase in Drosophila melanogaster

The relationship between the 50% survival time for flies feeding on a malathion-containing medium and the activity of acetylcholinesterase (AChE) was determined for 15 isofemale lines of Drosophila melanogaster. A significant correlation was found (r=0.28, P<0.05), with more resistant lines tending to have a lower level of AChE activity. An association between AChE and malathion resistance was also observed in a selection experiment. The AChE activity decreased in two of two populations selected for malathion resistance. AChE from these populations was altered in kinetic parameters (measured in crude head extracts) and electrophoretic mobility. Although the resistant AChE had a lower activity (V _m) on either a per milligram protein or a per individual basis, its apparent K _m for acetylthiocholine was lower than that of susceptible AChE. Recombination mapping of both low activity and fast electrophoretic mobility localized these traits to the region of the structural locus (Ace) on the third chromosome. The AChE activity of flies heterozygous for a variety of Ace lesions (kindly provided by Dr. W. M. Gelbart) was consistent with this location. The changes in AChE were suggested to have been caused by selection of alleles at the Ace locus.This work was supported by NSERC Grants A5857, G0183, and A0629.     

Characteristics of 3-O-methylfluorescein phosphate hydrolysis by the (Na+ + K+)-ATPase

With 3-O-methylfluorescein phosphate (3-OMFP) as substrate for the phosphatase reaction catalyzed by the (Na⁺ + K⁺)-ATPase, a number of properties of that reaction differ from those with the common substratep-nitrophenyl phosphate (NPP): theK _m is 2 orders of magnitude less and the V_max is two times greater, and dimethyl sulfoxide (Me₂SO) inhibits rather than stimulates. In addition, reducing the incubation pH decreases both theK _m and V_max for K⁺-activated 3-OMFP hydrolysis as well as theK _0.5 for K⁺ activation. However, reducing the incubation pH increases inhibition by P_i and the V_max for 3-OMFP hydrolysis in the absence of K⁺. When choline chloride is varied reciprocally with NaCl to maintain the ionic strength constant, NaCl inhibits K⁺-activated 3-OMFP hydrolysis modestly with 10 mM KCl, but stimulates (in the range 5–30 mM NaCl) with suboptimal (0.35 mM) KCl. In the absence of K⁺, however, NaCl stimulates increasingly over the range 5–100 mM when the ionic strength is held constant. These observations are interpreted in terms of (a) differential effects of the ligands on enzyme conformations; (b) alternative reaction pathways in the absence of Na⁺, with a faster, phosphorylating pathway more readily available to 3-OMFP than to NPP; and (c) a (Na⁺ + K⁺)-phosphatase pathway, most apparent at suboptimal K⁺ concentrations, that is also more readily available to 3-OMFP.Abbreviations Et₃N triethyl amine - FITC fluorescein isothiocyanate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - MES 2-(N-morpholino)ethanesulfonate - Me₂SO dimethyl sulfoxide - NPP p-nitrophenyl phosphate - 3-OMFP 3-O-methylfluorescein phosphate - TNP-ATP 2, (or 3)-O-(2,4,6-trinitrophenyl)-ATP     

Mode of action of furosemide on the chloride-dependent short-circuit current across the ciliary body epithelium of toad eyes

Summary The effects of furosemide on the chloride-dependent short-circuit current across the toad ciliary epithelium were examined. Under control conditions, the short-circuit current obeyed Michaelis-Menten kinetics against medium chloride concentration, the Michaelis constant (K _m ) for chloride being 90mm and the maximal short-circuit current (V _max) 128 A/cm². Furosemide added to the aqueous side of the epithelium rapidly reduced the short-circuit current; the effect was reversible. The effect of furosemide addition to the stromal side was much smaller and slower than that from the aqueous side. The dose-dependent range of furosemide action was from 0.1 m to 1mm with 50% inhibition occurring at about 3 m. Line-weaver-Burk plot of the short-circuit current against the chloride concentration showed that furosemide decreased the value ofV _max and increased theK _m ; the inhibition being of mixed type. A Hill plot of the dose-response curve yielding a slope of unity suggested one furosemide molecule combines with one chloride transport site. Probenecid, a competitive inhibitor of organic acid transport, reduced the effects of furosemide significantly when added simultaneously. The involvement of organic acid transport system in the mechanism of furosemide action on chloride transport was suggested.Department of Ophthalmology.     

Characterization of cholinesterase of muscularis muscle of Bufo marinus

We have characterized the cholinesterase (ChE) of muscularis muscle of Bufo marinus by selectively using specific inhibitors of acetylcholinesterase and pseudocholinesterase and observing susceptibility to inhibition when substrate is present in excess. The ChE activity in this preparation due to acetylcholinesterase (AChE) and pseudocholinesterase (BuChE) was 90 and 10%, respectively. The optimum temperature and pH for the ChE were 38 degrees C and 7.4, respectively and the excess substrate inhibition was noted above a pS of 2.6. The Km for acetylthiocholine (ASCh) was 0.76 X 10(-4) M.     

SUBSTRATE SPECIFICITY AND DEVELOPMENTAL CHANGES OF ACETYLCHOLINESTERASE IN COTTON BOLLWORM*

Abstract The substrate specificity and developmental changes of acetylcholinesterase (AChE) of cotton bollworm, Helicoverpa armigera Hübner, were investigated during 1991 to 1994. The insects were collected from Handan suburbs of Hebei Province and Guan County of Shandong Province. The results show that the specific activity and Michaelis constants (k_m) of AChE toward acetylthiocholine (ATCH) and acetyl-β-methyl-thiocholine (MeTCh) regularly varied with the developmental stage of cotton bollworm. The two peaks of the specific activity were observed respectively in the third instar and sixth instar of larvae. The specific activity of AChE in pupae was the lowest and that in heads of four-days moth was the highest in various developmental stages of cotton bollworms. The tendency of K_m and maximum velocity (V_max) was identical with the change of specific activity in the AChEs of cotton bollworm. The activation energy (Ea) of AChE toward MeTCh in pupae and adults was 3. 9–4. 3 times as much as that of, larvae in cotton bollworms from Handan of Hebei Province. It suggests that the spending energies of AChE for hydrolysing substrate are different in larva, pupa and adult. The optimum conditions of AChE reaction with ATCh in larvae were 50–100 mg of tissue weights for the amount of enzyme, 10–20 minutes for the reaction time, 35°C for the reaction temperature and 8. 0 for the reaction pH value.     

Kinetic Analyses of the Substrate Inhibition of <Emphasis Type="Italic">Paramecium</Emphasis> Arginine Kinase

Paramecium tetraurelia expresses four types of arginine kinase (AK1–AK4). In a previous study, we showed that AK3 is characterized by typical arginine substrate inhibition, where enzymatic activity markedly decreases near a concentration of 1 mM of arginine substrate. This is in sharp contrast to the three other AK types, which obey the Michaelis–Menten reaction curve. Since cellular arginine concentration in another ciliate Tetrahymena is estimated to be 3–15 mM in vivo, Paramecium AK3 likely functions in conditions that are strongly affected by substrate inhibition. The purpose of this work is to find some novel aspect on the kinetic mechanism of the substrate inhibition of Paramecium AK3 enzyme. Substrate inhibition kinetics for AK3 were analyzed using three models and their validity were evaluated with three static parameters (R², AICc, and Sy.x). The most accurate model indicated that not only ES but also the SES complex reacts to form products, the latter being the complex with two substrates in the active center. The maximum reaction rate for the SES complex, V_max^SES?=?30.4 µmol Pi/min/mg protein, was one-eighth of the ES complex, V_max^ES?=?241.7. The dissociation constant for the SES complex (K_i^SES: 0.34 mM) was two times smaller than that of the ES complex (K_s^ES: 0.61 mM), suggesting that after the primary binding of the arginine substrate (ES complex formation), the binding of a second arginine to the secondarily induced inhibitory site is accelerated to form an SES complex with a lower V_max^SES. The same kinetics were used for the S79A, S80A, and V81A mutants. The results indicate that the S79 residue is significantly involved in the process of binding the second arginine substrate. Herein, the K_i^SES value was ten times (3.62 mM) the value for the wild-type (0.34 mM), weakening substrate inhibition. In contrast, V_max^ES and V_max^SES values for the mutants decreased by one-third, except for the V_max^SES of the S79A mutant, which had a value that was comparable with the value for the wild-type.