The antienzymic activities of 14 organophosphorous compounds, the derivatives of dialkyl thiophosphoric acid, towards the acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and carboxylesterase (CE) from the spring grain aphid and mammals were investigated. The dependence of inhibitory activity of the compounds on their alkyl radical length was shown to be different for the AchE from the aphid and man. Some less pronounced differences in this dependence were revealed between the BuChEs from the aphid and horse as well as between the CEs from the aphid, mouse and red spider mite. The data give evidence of a distinction in structure of the active surfaces of the enzymes from the aphid and mammals. Some peculiar properties of the aphid cholinesterases are discussed taking account of the results of the present and previous papers.
The properties of aminostigmine in comparison with those of other carbamate inhibitors of cholinesterases have been studied in vitro using potentiometric titration and Ellman methods. The bimolecular constants of the inhibition rate of acetyl-, butyryl- and propionylcholinesterase were found to be equal to (8.0-14.0).10(5) (3.8-7.7).10(5) and 11.0.10(5) M-1.min-1, respectively. In terms of inhibitory activity, aminostrigmine is comparable to neostigmine methylsulphate, being inferior to physostigmine and superior to pyridistigmine. The rate of decarbamylation of acetylcholinesterase inhibited by aminostigmine measured by the dilution method, by creating excessive acetylcholine and by dialysis is characterized by k2c constants equal to (1.1-1.6).10(-2), (2.5-2.8).10(-2) and 0.025.10(-2) min-1, respectively. On the whole, aminostigmine belongs to slowly reversible inhibitors. Being carbamylated by aminostigmine, the enzyme is resistant to reactivation by TMB-4(Trimedoxime) and HI-6. At (4-6).10(-7) M aminostigmine prevents by 50% the irreversible binding of cholinesterase by certain organophosphate inhibitors of cholinesterase when the latter are used at concentrations needed to inhibit the enzymatic activity by 85-90%.
Title: [The effect of pH and reversible inhibitors on decarbamylation of acetylcholinesterase] Kugusheva LI, Rozengart VI, Sherstobitov OE Ref: Ukr Biokhim Zh (1978), 63:29, 1991 : PubMed
Decarbamylation rate of membrane-bound methyl- and dimethyl-carbamylated acetylcholinesterase of human erythrocytes and bovine brain is reliably 1.1-1.6 times lower than that of the soluble enzyme. Such reversible inhibitors as tacrine (of non-competition action), ambenonium (mixed action) and galanthamine (competitive type of action) decelerate the decarbamylation rate of acetylcholinesterase. At pH 6 tacrine inhibits the reduction rate of soluble acetylcholinesterase activity of human erythrocytes more intensively than that of membrane-bound acetylcholinesterase. No differences in decarbamylation rate were found for the both forms of the enzyme at pH 8. Tacrine, a non-competitive inhibitor in concentrations below the inhibition constant (Ki = 1.4 x 10(-7) M) exerts the most intensive effect on the decarbamylation rate of methyl- and dimethylcarbamylated acetylcholinesterase of the mouse brain, while ambenonium and galanthamine in concentrations much (tens times) exceeding their Ki (3.1 x 10(-10) M and 4.4 x 10(-7) M, respectively) provide a decrease of the decarbamylation rate.
Title: Poster: The effect of conformation isomerism and hydrophobicity of organophosphates on their interaction with cholinesterases of different origin Vasiljeva TI, Kugusheva LI, Rozengart VI Ref: In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology, (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC:278, 1991 : PubMed
The soluble and immobilized cholinesterases (acetyl cholinesterase of human blood erythrocytes (EC 3.1.1.7) and butyryl cholinesterase of equine blood serum, (EC 3.1.1.8] were inactivated by such irreversible inhibitors as diisopropyl fluorophosphate (DFP), O,O-dimethyl-O-(2,2)-dichlorovinyl) phosphate (DDVP), paraoxone, armine. The inactivated enzymes were reactivated under the effect of TMB-4(Trimedoxime) (1,1'-trimethylene-bis)-4-formyl-pyridine bromide (dioxime). The values of the reactivation rate constants proved to be equal both for the soluble and immobilized cholinesterases inactivated by the same irreversible inhibitor. The immobilized enzyme is simpler and more correct to study the reactivating action than the soluble one.
Catalytic properties of human blood erythrocyte acetylcholinesterase and horse blood serum butyrylcholinesterase immobilized and nonimmobilized in the gelatin membrane have been comparatively studied. Cholinesterase immobilization induces an increase in the Michaelis constant value and a decrease in the maximum rate value in reactions of enzymic hydrolysis of thiocholine ethers, but exerts no effect on these kinetic parameters in case of enzymic hydrolysis of indophenylacetate. The effect of reversible inhibitors: galanthamine, N-methyl-4-piperidinyl benzylate and 1,2,3,4-tetrahydro-9-aminoacridine (tacrine), as well as of irreversible inhibitors: O-ethyl-O-(4-nitrophenyl)ethyl phosphonate (armin), diisopropyl fluorophosphate (DFP), O,O-diethyl-O-(4-nitrophenyl) phosphate (paraoxon) and O,O-dimethyl-O-(2,2-dichlorovinyl) phosphate (DDVP) on immobilized cholinesterases is weaker as compared with the effect on nonimmobilized enzymes. The results obtained are discussed for the effect of immobilization on the catalytically active enzyme surface.
Title: [Interaction of membrane-bound and solubilized acetylcholinesterase from human and bovine erythrocytes with organophosphorus inhibitors]. [Russian] Kugusheva LI, Rozengart VI Ref: Ukrainskii Biokhimicheskii Zhurnal, 58:13, 1986 : PubMed
Differences are found between the membrane-bound and soluble acetylcholinesterases of human and bovine erythrocytes when the enzyme interacts with organophosphoric inhibitors in the presence of acetylc choline and galantamine, a reverse inhibitor of acetylcholinesterase. In most cases prevention of inhibition of the soluble enzyme activity necessitates a higher (2-3 times higher) concentration of the protecting agent than protection of the membrane-bound enzyme. Concentrations of acetylcholine and galantamine providing a 50% protection of the enzyme did not practically depend on the strength of the anticholinesterase action of organophosphoric inhibitors.
Studies have been made on the effect of organophosphorus inhibitors on cholinesterases (CHe) from mammals (human and rabbit erythrocytes, mice brain) and arthropods (fly, spring grain aphid, rice weevil and spider tick). Organophosphorus inhibitors were presented by dialkylthiophosphates which contained normal or branching alkyls in the phosphoryl part of their molecule and lupinine or epilupinine residue in the other part of the latter. Increasing the length of alkyl in lupinine derivatives increased their inhibitory effect with respect to mammalian CHe, but decreased it in relation to all the investigated arthropod CHe. For epilupinine derivatives, the same relationship was found with mammalian CHe, being absent in arthropod enzymes. These data indicate the existence of significant differences in spatial structure of both esterase and anionic centers of the enzyme from mammals and arthropods. In arthropod CHe, hydrophobic regions of the esterase center are less developed that in mammalian CHe. The distance between the anionic and esterase centers of CHe is presumably different in the enzymes from different species.
