Cell-bound cholinesterase inTrichoderma harzianum

Cell-bound cholinesterase enzyme activity is reported for the first time in the mycelium ofTrichoderma harzianum. This enzyme hydrolyzes both the acetylcholine and the butyryl thiocholine esters. TheK _m andV _max for choline ester are 0.69 mM and 1.0 nmol acid released min^–1 g^–1 protein. However, the thiocholine ester has aK _m value of 2.2 mM andV _max value of 3.33 nmol product formed min.^–1 g^–1 protein. The enzyme is inhibited by eserine, a true classical cholinesterase inhibitor.     

Inhibition of cholinesterase by dialkylcarbamates

The pI50 index and separation coefficients of chosen 3-N,N-diethylaminophenyl-N',N'-dialkylcarbamates were determined. Index pL50 (pI50 = negative logarithm of molar concentration of inhibitor inhibiting the enzyme activity by 50%) describes the effectiveness of the inhibitor. The rate of ability of the inhibitor to pass the blood-brain barrier is usually described by the separation coefficient in a system n-octanol/water (K(ow)). Obtained results were compared with pL50 and K(ow) of Exelon, the commercially used drug against the Alzheimer's disease.     

Localization of cholinesterase activity in house-fly thoraces: Inhibition of cholinesterase with organophosphate compounds

Cholinesterase (ChE) activity was localized by a histochemical method in thoraces of non-treated, and tepp- and dicrotophos-(Bidrin^®)-treated houseflies. The findings indicate a good correlation between intoxication symptoms (knock-down) and inhibition of ChE activity in the cell body region rather than the neuropile or synaptic region of the compound ganglion. Results of histochemical studies agree well with quantitative pH-stat assays which show wide differences in levels of inhibition of total ChE activity in tepp- and dicrotophos-treated flies at knock-down.In contrast to 2 ChE's in mammalian brain, histochemical evidence is presented to support the conclusion of previous workers that a single ChE in houseflies hydrolyzes both acetylcholine and butyrylcholine.

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Zusammenfassung Die Cholinesterase (ChE)-Aktivität im Thorax von unbehandelten und mit Tepp-(Tetraäthylpyrophosphat) und Dicrotophos- ( Isomer von Dimethylphosphateester mit (E)-3-hydroxy-N, N-dimethylcrotoamide) (Bidrin) behandelten Stubenfliegen wurde mit einer histochemischen Methode lokalisiert. Die Ergebnisse deuten eine gute Korrelation zwischen Vergiftungserscheinungen (knock-down) und Hemmungen der ChE-Aktivität in der Region der Zellkörper an, aber nicht im Nerven bündel oder in der synaptischen Region des zusammengesetzten Ganglions. Resultate dieser histochemischen Versuche stimmen ziemlich gut überein mit den quantitativen pH-stat-Proben. Diese Proben zeigten einen weiten Unterschied in den Hemmungsgraden der totalen ChE-Aktivität von Tepp-und Dicrotophosbehandelten Fliegen während der Vergiftung.In Bestätigung der Schlußfolgerung früherer Bearbeiter beweist die histochemische Untersuchung, daß im Gegensatz zu zwei Cholinesterasen im Säugetiergehirn in der Stubenfliege eine einzige Cholinesterase Acetylcholin und Butyrylcholin hydrolisiert.

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This investigation was supported by National Institutes of Health Research Grant No. 5 ROI ES00253. Journal paper no. 743, University of Georgia College of Agriculture Experiment Stations, College Station, Athens.     

Structure of human serum cholinesterase

Human cholinesterase has recently been sequenced and cloned. It is a glycoprotein of 4 identical subunits, each subunit containing 9 carbohydrate chains and 3.5 disulfide bonds. Protein folding is likely to be very similar in human cholinesterase and Torpedo acetylcholinesterase. The cholinesterases have no significant sequence homology with the serine proteases and seem to belong to a separate serine esterase family.     

Dibucaine inhibition of serum cholinesterase

The dibucaine number (DN) was determined for serum cholinesterase (EC 3.1.1.8, SChE) in plasma samples. The ones with a DN of 79-82 were used, because they had the "usual" SChE variant. The enzyme was assayed colorimetrically by the reaction of 5,5'-dithiobis-[2-nitrobenzoic acid] (DTNB) with the free sulfhydryl groups of thiocholine that were produced by the enzyme reaction with butrylthiocholine (BuTch) or acetylthiocholine (AcTch) substrates, and measured at 412 nm. Dibucaine, a quaternary ammonium compound, inhibited SChE to a minimum within 2 min in a reversible manner. The inhibition was very potent. It had an IC(50) of 5.3 microM with BuTch or 3.8 microM with AcTch. The inhibition was competitive with respect to BuTch with a K(i) of 1.3 microM and a linear-mixed type (competitive/noncompetitive) with respect to AcTch with inhibition constants, K(i) and K(I) of 0.66 and 2.5 microM, respectively. Dibucaine possesses a butoxy side chain that is similar to the butryl group of BuTch and longer by an ethylene group from AcTch. This may account for the difference in inhibition behavior. It may also suggest the existence of an additional binding site, other than the anionic binding site, and of a hydrophobic nature.     

Stereochemical aspects of cholinesterase catalysis

Proceeding from the sterochemical regularities of the nucleophilic substitution reaction at the carbonyl group and the assumption that the spatial structure of the active center of cholinesterases is complementary to the molecule of the ester substrates for these enzymes, some general features of the stereoselectivity phenomena in the reactions of acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) with organophosphorus inhibitors are discussed. For these enzymes the models of the active center are proposed in terms of different binding sites and the catalytic center. On the basis of this model, the stereochemical pecularities and the physicochemical background of the stereoselectivity effects on enzyme inhibition, reactivation, and “aging” reactions can be understood. Knowledge of the absolute configuration of several chiral organophosphorus inhibitors also makes it possible to determine the absolute spatial arrangement of the hydrophobic binding sites on the active surface of cholinesterases.