Vera Simeon

Mechanism of inhibition in vitro of mammalian acetylcholinesterase and cholinesterase in solutions of O,O-dimethyl 2,2,2-trichloro-1-hydroxyethyl phosphonate (trichlorphon)

Abstract The rate of decomposition of trichlorphon into DDVP was measured polarographically at pH 7.4. The first order rate constants of decomposition at 25° are 7.27 × 10 −4 and 6.05 × 10 −4 min −1 for trichlorphon concentrations of 0.150 and 15.0 mM respectively; at 37° the corresponding rate constants are 53.1 × 10 −4 and 37.1 × 10 −4 min −1 . The rate of decomposition of trichlorphon was also calculated from the kinetics of inhibition of acetylcholinesterase (EC 3.1.1.7) and cholinesterase (EC 3.1.1.8) in trichlorphon solutions at 25° and 37° (pH 7.4). The following enzyme sources were used: bovine erythrocytes and rat brain acetylcholinesterase, and human, horse and rat plasma cholinesterase. The rate of decomposition of trichlorphon was calculated by assuming that only DDVP formed from trichlorphon is the enzyme inhibitor, while trichlorphon itself does not act as an inhibitor. The calculated rate constants for the decomposition of trichlorphon are lower or just within the range of the rate constants obtained by the polarographic method. This agreement was taken as kinetic evidence that trichlorphon is not an inhibitor of mammalian cholinesterases. The effect of pH on enzyme inhibition supports this conclusion. The rate of inhibition of bovine erythrocyte acetylcholinesterase by DDVP is the same at pH 7.4 and pH 6.0 (37°). However, the rate of enzyme inhibition in trichlorphon solutions is 30 times faster at pH 7.4 than at pH 6.0, and this agrees with the greater stability of trichlorphon at the lower pH value. The rate of spontaneous reactivation of the enzyme was measured (37°, pH 7.4) after inhibition in trichlorphon solutions of acetylcholinesterase (human and bovine erythrocytes) and cholinesterase (human plasma). For all three enzyme preparations, the rate of spontaneous reactivation was the same as that obtained after inhibition by DDVP. All results point to the conclusion that trichlorphon in vitro is not an inhibitor of mammalian cholinesterases.

Serum paraoxonase and cholineseterase activities in individuals with lipid and glucose metabolism disorders

In patients with hyperlipaemia, serum paraoxonase activities were polymodally distributed with 75% individuals in the low activity mode. In the same patients the distribution of serum cholinesterase activities was unimodal, but asymmetrical. Patients with impaired glucose tolerance or non-insulin-dependent diabetes mellitus had slightly higher cholinesterase activities than patients with hyperlipaemia only.

Binding sites on acetylcholinesterase for reversible ligands and phosphorylating agents: A theoretical model tested on haloxon and phosphostigmine

The reaction of acetylcholinesterase (EC 3.1.1.7; human erythrocytes) with phosphostigmine, haloxon and VX was studied, and the effect of three reversible ligands (TMA, edrophonium, coumarin) and of acetylthiocholine upon the time-dependent and time-independent (reversible) inhibition by the organophosphates was evaluated. The three ligands and acetylthiocholine decreased the second-order rate constant of phosphorylation by a factor proportional to the enzyme-ligand dissociation constant, or to both Km and Kss (Michaelis constant and the substrate-inhibition constant for acetylthiocholine) irrespective of the organophosphate. However, the time-independent inhibitions by phosphostigmine and haloxon were differently affected. Acetylthiocholine affected the time-independent inhibition by phosphostigmine by a factor proportional to Km, and that by haloxon by a factor proportional to Kss. Coumarin had no effect on the time-independent inhibition by phosphostigmine, while TMA and edrophonium displaced phosphostigmine from its complex. Coumarin displaced haloxon from its complex with the enzyme, while TMA and edrophonium had no effect. We conclude that phosphostigmine and haloxon bind reversibly to different sites on the enzyme and the experiments agree with a theoretical model that haloxon binds reversibly to a peripheral site on acetylcholinesterase, and phosphostigmine to the catalytic site.

Differentiation of esterases reacting with organophosphorus compounds.

The hydrolysis of paraoxon (POX), phenylacetate (PA) and beta-naphthylacetate (BNA) was studied in human serum. Based upon correlations between enzyme activities, upon reversible inhibition by EDTA and upon progressive inhibition by iso-OMPA, tabun, eserine and bis-4 nitrophenylphosphate, the following conclusions were drawn about the number and specificity of enzymes involved in the hydrolysis. Two paraxonases hydrolyse paraoxon: one sensitive and the other insensitive to EDTA. The EDTA-sensitive paraoxonase also hydrolysed BNA. The EDTA-insensitive hydrolysis of BNA and PA was attributed to a serine esterase. The EDTA-sensitive hydrolysis of PA is probably due to more than one enzyme, which might be an arylesterase and a carboxylesterase.

Cholinesterases in rabbit serum

1. Rabbit serum was shown to contain two cholinesterases which hydrolysed acetylthiocholine and butyrylthiocholine and one cholinesterase which hydrolysed only butyrylthiocholine. 2. The three enzymes were identified by the kinetics of heat inactivation and kinetics of phosphorylation by the organophosphate VX. 3. Using selective inhibitors (iso-OMPA, eserine, BNPP and BW-284C51) it was shown that the hydrolysis of acetylthiocholine and butyrylthiocholine in untreated native serum had properties of acetylcholinesterase (EC 3.1.1.7), butyrylcholinesterase (EC 3.1.1.8) and also some properties of carboxylesterase (EC 3.1.1.1). 4. Separation of proteins (on PAA-gels) in untreated native serum gave four bands with acetylthiocholine and three with butyrylthiocholine. 5. The two cholinesterases hydrolysing both substrates corresponded to the slow moving bands on the gel. 6. The fastest moving band hydrolysing only butyrylthiocholine could be attributed to the cholinesterase least sensitive to VX.

Reactions of Usual and Atypical Human Serum Cholinesterase Phenotypes with Progressive and Reversible Inhibitors

AbstractThe reaction of usual (U) and atypical (A) cholinesterase phenotypes was studied with six organophosphorus compounds, two pyridinium oximes (HI-6 and PAM-2) and with 4–4-bipyridine (4,4-BP). No difference in the inhibition rate constants for the two phenotypes was found with the progressive inhibitors tabun, sarin, paraoxon and soman. The other two progressive inhibitors, VX and the positively charged phosphostigmine, inhibited the U phenotype more strongly than the A phenotype.The positively charged reversible inhibitor HI-6 showed a higher affinity for the U than for the A phenotype, while PAM-2 and the non-charged 4,4′-BP did not show a significant difference in their affinity towards the two enzymes.Both phenotypes phosphylated by VX or sarin were reactivatable by HI-6 and PAM-2, and the A phenotype was always reactivated more slowly than the U phenotype. The paraoxon-inhibited phenotypes were reactivated at equal rates with PAM-2 but were not reactivated with HI-6. The phosphylated phenotypes...

Heat inactivation of paraoxonase and arylesterase activities in human and rabbit serum

The heat inactivation of esterases in human and rabbit serum was followed at 50 and 55 degrees C by measuring the decrease of activity with paraoxon, phenylacetate and beta-naphthylacetate as substrates. The rate of inactivation measured with the three substrates was slightly, but significantly different, indicating that the substrates are hydrolysed by different enzymes.

Kinetics of the inhibition of human serum cholinesterase phenotypes with the dimethylcarbamate of (2-hydroxy-5-phenylbenzyl)-trimethylammonium bromide (Ro 02-0683)

The inhibition of the human serum cholinesterase phenotypes, usual (U), atypical (A) and heterozygous (UA), by the dimethylcarbamate of (2-hydroxy-5-phenylbenzyl)-trimethylammonium bromide (Ro 02-0683), was followed with benzoylcholine, acetyl-, butyryl- and propionyl-thiocholine as substrates. The first-order rate constants were calculated from the linear part of the inhibition curves and were independent of the substrate used for measuring the enzyme activity. The second-order rate constants for the U, UA and A phenotypes were 8.3 x 10(6), 6.1 x 10(6) and 0.05 x 10(6) M-1 min-1, respectively. The constant of the enzyme-inhibitor complex for the atypical serum was 7.7 microM, and the rate of carbamylation of the enzyme was 0.386 min-1. The rate of reactivation of carbamylated usual and atypical enzyme was found to be same; the half-time of reactivation was about 3.5 hr. The deviation from the linearity of the inhibition course was explained by spontaneous reactivation of the inhibited enzyme; the theoretical inhibition curves were in good agreement with the experimentally obtained values. The three phenotypes could be distinguished by the rate of inhibition by the dimethylcarbamate, Ro 02-0683, in the progressive phase of inhibition or by the degree of inhibition in the apparent steady-state.

Catalytic Properties and Classification of Phosphoric Triester Hydrolases: Edta-Insensitive and Edta-Sensitive Paraoxonases in Sera of Non-Diseased and Diseased Population Groups

Phosphoric triester hydrolases (EC 3.1.8) are defined as enzymes acting on organophosphorus compounds, including esters of phosphonic and phosphinic acids, and on phosphorus anhydride bonds; the enzymes are subdivided into paraoxonase (EC 3.1.8.1) and DFP-ase (EC 3.1.8.2) [1].

Kinetics and Mechanisms of Reversible and Progressive Inhibition of Acetylcholinesterase

This paper concerns the interaction of reversible ligands, particularly oximes, with acetylcholinesterase (AChE) and their effect upon the catalytic properties of the enzyme.