Cholinesterase and phenyl valerate-esterase activities sensitive to organophosphorus compounds in membranes of chicken brain.

Some effects of organophosphorus compounds (OPs) esters cannot be explained by action on currently recognized targets acetylcholinesterase or neuropathy target esterase (NTE). In previous studies, in membrane chicken brain fractions, four components (EPα, EPß, EPγ and EPδ) of phenyl valerate esterase activity (PVase) had been kinetically discriminated combining data of several inhibitors (paraoxon, mipafox, PMSF). EPγ is belonging to NTE. The relationship of PVase components and acetylcholine-hydrolyzing activity (cholinesterase activity) is studied herein. Only EPα PVase activity showed inhibition in the presence of acetylthiocholine, similarly to a non-competitive model. EPα is highly sensitive to mipafox and paraoxon, but is resistant to PMSF, and is spontaneously reactivated when inhibited with paraoxon. In this papers we shows that cholinesterase activities showed inhibition kinetic by PV, which does not fit with a competitive inhibition model when tested for the same experimental conditions used to discriminate the PVase components. Four enzymatic components (CP1, CP2, CP3 and CP4) were discriminated in cholinesterase activity in the membrane fraction according to their sensitivity to irreversible inhibitors mipafox, paraoxon, PMSF and iso-OMPA. Components CP1 and CP2 could be related to EPα as they showed interactions between substrates and similar inhibitory kinetic properties to the tested inhibitors.

Acetylcholine-hydrolyzing activities in soluble brain fraction: Characterization with reversible and irreversible inhibitors.

Some effects of organophosphorus compounds (OPs) esters cannot be explained through actions on currently recognized targets acetylcholinesterase or neuropathy target esterase (NTE). In soluble chicken brain fraction, three components (Eα, Eß and Eγ) of pheny lvalerate esterase activity (PVase) were kinetically discriminated and their relationship with acetylcholine-hydrolyzing activity (cholinesterase activity) were studied in previous works. In this work, four enzymatic components (CS1, CS2, CS3 and CS4) of cholinesterase activity have been discriminated in soluble fraction, according to their sensitivity to irreversible inhibitors mipafox, paraoxon, PMSF and iso-OMPA and to reversible inhibitors ethopropazine and BW284C51. Cholinesterase component CS1 can be related to the Eα component of PVase activity and identified as butyrylcholinesterase (BuChE). No association and similarities can be stablished among the other PVase component (Eß and Eγ) with the other cholinesterase components (CS2, CS3, CS4). The kinetic analysis has allowed us to stablish a method for discriminating the enzymatic component based on a simple test with two inhibitors. It can be used as biomarker in toxicological studies and for monitoring these cholinesterase components during isolation and molecular identification processes, which will allow OP toxicity to be understood by a multi-target approach.

Interaction between substrates suggests a relationship between organophosphorus-sensitive phenylvalerate- and acetylcholine-hydrolyzing activities in chicken brain.

Organophosphorus compounds (OPs) induce neurotoxic disorders through interactions with well-known target esterases, such as acetylcholinesterase and neuropathy target esterase (NTE). However, OPs interact with other esterases of unknown biological function. In soluble chicken brain fractions, three components of enzymatic phenylvalerate esterase activity (PVase) called Eα, Eß and Eγ, have been kinetically discriminated. These components are studied in this work for the relationship with acetylcholine-hydrolyzing activity. When Eα PVase activity (resistant PVase activity to 1500 µM PMSF for 30 min) was tested with different acetylthiocholine concentrations, inhibition was observed. The best-fitting model to the data was the non-competitive inhibition model (Km=0.12, 0.22 mM, Ki=6.6, 7.6 mM). Resistant acetylthiocholine-hydrolyzing activity to 1500 µM PMSF was inhibited by phenylvalerate showing competitive inhibition (Km=0.09, 0.11 mM; Ki=1.7, 2.2 mM). Eß PVase activity (resistant PVase activity to 25 µM mipafox for 30 min) was not affected by the presence of acetylthiocholine, while resistant acetylthiocholine-hydrolyzing activity to 25 µM mipafox showed competitive inhibition in the presence of phenylvalerate (Km=0.05, 0.06 mM; Ki=0.44, 0.58 mM). The interactions observed between the substrates of AChE and PVase suggest that part of PVase activity might be a protein with acetylthiocholine-hydrolyzing activity.

Cholinesterase assay by an efficient fixed time endpoint method.

Many cholinesterase assays are performed to study the inhibition of cholinesterase (ChE) activity. Frequently a large number of samples are processed and Ellman's method [1] is the most commonly used [2,3]. Activity is estimated from the increment in absorbance between two reaction times when the reaction is not stopped. Bellino et al. [4] described a method based on Ellman's method whereby the reaction was stopped with SDS and then the absorbance was measured. In these methods, the chromogen reagent 5,5'dithiobis nitro benzoic acid (DTNB) is added with the substrate and colour is monitored. Some authors pointed that the chromogen can alter cholinesterase activity [5].•A modification of Bellino's method is proposed for acetylcholine-hydrolyzing activity determinations that is based on stopping the reaction after a fixed substrate reaction time using a mixture of detergent SDS and DTNB.•The method may be adapted to the user needs by modifying the enzyme concentration and applied for simultaneously testing many samples in parallel; i.e. for complex experiments of kinetics assays with organophosphate inhibitors in different tissues.

Rabbit serum albumin hydrolyzes the carbamate carbaryl.

One of the main detoxification processes of the carbamate insecticides is the hydrolysis of the carbamic ester bond. Carboxylesterases seem to play important roles in the metabolization of carbamates. This study performs a biochemical characterization of the capabilities of rabbit serum albumin (RSA) to hydrolyze the carbamate carbaryl. Rabbit serum albumin was able to hydrolyze carbaryl with a K(cat) of 7.1 x 10(-5) s(-1). The K(m) for this hydrolysis reaction was 240 microM. Human, chicken, and bovine serum albumins were also able to hydrolyze carbaryl. The divalent cation Cu(2+) at 1 mM concentration inhibited around 50% of the hydrolysis of carbaryl by RSA. Other mono- and divalent cations at 1 mM concentration and 5 mM EDTA exerted no significant effects on the hydrolysis of carbaryl by RSA. The inhibition of the carbaryl hydrolysis by sulfydril blocking agents suggests that a cysteine residue plays an important role in the active center of the catalytic activity. Both caprylic and palmitic acids were noncompetitive inhibitors of the carbaryl hydrolysis by RSA. The carboxyl ester p-nitrophenyl butyrate is a substrate of RSA and competitively inhibited the hydrolysis of carbaryl by this protein, suggesting that the hydrolysis of carbaryl and the hydrolysis of carboxyl esters occur in the same catalytic site and through a similar mechanism. This mechanism might be based on the carbamylation of a tyrosine residue of the RSA. Serum albumin is a protein universally present in nontarget species of insecticides; therefore, the capability of this protein to hydrolyze other carbamates must be studied because it might have important toxicological and ecotoxicological implications.