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.     

Phenylethanolamine N-methyltransferase inhibition: re-evaluation of kinetic data

Inhibitors of phenylethanolamine N-methyltransferase [PNMT, the enzyme that catalyzes the final step in the biosynthesis of epinephrine (Epi)] may be of use in determining the role of Epi in the central nervous system. Here we demonstrate that a routinely used assay for screening PNMT inhibitors is not appropriate for those inhibitors having K(i) values less than 1 microM. A revised assay has been developed that shows some inhibitors bind two orders of magnitude more tightly than previously reported.     

Analysis of kinetic data for irreversible enzyme inhibition.

Many organophosphorus compounds are irreversible inhibitors of acetylcholinesterase. The methods used in the literature to determine the inhibition kinetic constants usually involve either manual determination of the slope at various points along the inhibition progress curve or fitting polynomials to the curve. The present study investigates the use of non-linear-regression analysis to determine the various parameters. A method is suggested that yields accurate values for the inhibition constants under a range of circumstances.     

The inhibition and reactivation of human maternal and fetal plasma cholinesterase following exposure to the organophosphate, dichlorvos.

Spontaneous and chemically-induced reactivation of organophosphate-inhibited cholinesterase were studied using as an enzyme source plasma obtained from non-pregnant females, pregnant females at term and their respective neonates, sampled immediately following delivery. Aliquots of plasma were incubated with dichlorvos (10^?6M) for 5 min at 37°C resulting in a 96 percent inhibition of cholinesterase activity in all three groups at which time either pralidoxime chloride (10^?3M) or an equivalent volume of saline was added to the reaction flask and the restoration of cholinesterase activity was monitored over the next 120 min. Pralidoxime-mediated cholinesterase reactivation in ‘non-pregnancy’ plasma was significantly greater than that observed in either ‘maternal’ or ‘fetal’ plasma, however, no significant difference was noted in reactivation rates for these latter two groups. Significant differences were also observed in the rates of spontaneous reactivation, however, after correcting for this, there were still significant differences in the rates of pralidoxime-mediated reactivation (non-pregnant > pregnant ≥ fetal).     

Calibration of bioaffinity assays using kinetic data

Bioaffinity assays are usually calibrated by using a set of standard measurements fitted to a simple empirical model. In this paper, a new calibration approach based on mechanistic model of reaction kinetics is presented. When the calibration assay is known in terms of reaction mechanism, incubation time, initial concentration, and rate constants, one can back-calculate concentrations of unknown samples measured in a nonequilibrium time point. This paper describes a calculation method of unknown sample concentrations based on kinetically measured single calibration assay point. The theoretical results are verified by two common in-vitro diagnostic assays.     

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.     

The two 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase isoenzymes from Saccharomyces cerevisiae show different kinetic modes of inhibition

Activity of the tyrosine-inhibitable 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (EC 4.1.2.15) from Saccharomyces cerevisiae that was encoded by the ARO4 gene cloned on a high-copy-number plasmid was enhanced 64-fold as compared to the wild-type. The enzyme was purified to apparent homogeneity from the strain that harbored this recombinant plasmid. The estimated molecular weight of 42,000 of the enzyme corresponded to the calculated molecular mass of 40 kDa deduced from the DNA sequence. The enzyme could be inactivated by EDTA in a reaction that was reversed by several bivalent metal ions; presumably a metal cofactor is required for enzymatic catalysis. The Michaelis constant of the enzyme was 125 μM for phosphoenolpyruvate and 500 μM for erythrose 4-phosphate. The rate constant was calculated as 6 s^–1, and kinetic data indicated a sequential mechanism of the enzymatic reaction. Tyrosine was a competitive inhibitor with phosphoenolpyruvate as substrate of the enzyme (K _i of 0.9 μM) and a noncompetitive inhibitor with erythrose 4-phosphate as substrate. This is in contrast to the ARO3-encoded isoenzyme, where phenylalanine is a competitive inhibitor with erythrose 4-phosphate as a substrate of the enzyme and a noncompetitive inhibitor with phosphoenolpyruvate as substrate. Received: 29 December 1997 / Accepted: 3 March 1998     

Activity, isoenzymes and purity of mushroom tyrosinase in commercial preparations

Several lots of commercial tyrosinase preparations were examined with regard to their enzyme activity, isoenzyme composition and purity. Enzyme activity toward catechol, -dopa and tyrosine showed significant variations from lot to lot and activation by SDS. Distribution of isoenzyme forms also varied from lot to lot. Comparisons of electrophoretic and isoelectric focusing protein profiles showed considerable differences and distributions of the proteins in each sample. Tyrosinase appeared to be a minor component in each preparation when compared to a partially purified enzyme. Investigators using commercial tyrosinase should exercise caution in interpreting data due to the presence of different isoenzyme forms, their distribution in various lots, and the presence of numerous other proteins.     

Determination of the kinetic constants of inhibited acetyl cholinesterase

A method for the determination of kinetic constants of inhibited acetylcholinesterase, in the presence of substrate, is proposed. Theoretical, experimental and data treatment aspects are described for a suitable analysis of inhibitors acting with one or two steps. Limitations of other methods recently reported are pointed out.     

On the inhibition of cholinesterase by D-tubocurarine

Some investigation in this laboratory pointed to an unexpectedly slow inhibition of cholinesterase by D-tubocurarine, occurring in addition to a typically instantaneous inhibition. In order to elucidate this phenomenon, the hydrolysis of butyrylthiocholine catalyzed by cholinesterase was recorded, in the absence and presence of D-tubocurarine, on a stopped-flow apparatus. Experimental results were analyzed by classical kinetic methods and by means of mathematical modeling. It was found that the inhibition is of a double character, consisting of an instantaneous phase and a slow one occurring in a minute time scale. It seems that the action of D-tubocurarine is a consequence of an instantaneous binding of D-tubocurarine to a peripheral site, followed by a relatively slow conformational transition in the enzyme.     

Comparative studies on kinetic behavior of horseradish peroxidase isoenzymes

Kinetic parameters for each reaction step of the peroxidase-catalyzed reaction were determined by the stopped-flow technique on three distinct isoenzymes: acidic A2, neutral C1, and basic E5. The pH dependence of the reaction for the formation of compound I with hydrogen peroxide was examined. The three isoenzymes had a common ionizing group at about pK 4 which affects the rate constant for the formation of compound I. The heat of ionization determined from the temperature dependence of the dissociation constant of the group strongly suggested that it is of carboxyl nature. The rate constant for isoenzyme A2 was a tenth of those for the other two isoenzymes over the whole range of pH. Furthermore, the thermodynamic parameters of isoenzyme A2 were found to be different from those for the other two isoenzymes. These difference as well as the different behavior in alkaline transition of the isoenzymes are discussed in relation to the sixth ligand of the heme. The rate constant of the reduction of compound I and compound II by ferrocyanide were also determined. In both reduction steps, the pH profiles of the apparent rate constant for isoenzyme A2 and E5 were similar, but they were different from that of C1. The ionization with pK 5.29, which was detected only in isoenzyme C1, may be attributed to a group near the porphyrin ring as a stabilizer for the pi-cation radical.     

Identification of the root of Punica granatum in galenic preparations using TLC

The alkaloids present in the root of "Punica granatum" have been extracted by two different methods: extraction by Soxlet and extraction by steam distillation. Then the extracts have been compared by TLC chromatography using different solvents and specific chromogen reagents. The presence of pseudo-pelletierine has been confirmed in both the extractive solution by reaction with conc. K2Cr2O7. The above results explains the toxic activity of the unsuitable galenic preparations.