Title: Mipafox differential inhibition assay for heart muscle cholinesterases: substrate specificity and inhibition of three isoenzymes by physostigmine and quinidine Chemnitius JM, Haselmeyer KH, Gonska BD, Kreuzer H, Zech R Ref: General Pharmacology, 28:567, 1997 : PubMed
1. A differential inhibition assay was developed for the quantitative determination of cholinesterase isoenzymes acetylcholinesterase (AChE; EC 3.1.1.7), cholinesterase (BChE; EC 3.1.1.8), and atypical cholinesterase in small samples of left ventricular porcine heart muscle. 2. The assay is based on kinetic analysis of irreversible cholinesterase inhibition by the organophosphorus compound N,N'-di-isopropylphosphorodiamidic fluoride (mipafox). With acetylthiocholine (ASCh) as substrate (1.25 mM), hydrolytic activities (A) of cholinesterase isoenzymes were determined after preincubation (60 min, 25 degrees C) of heart muscle samples with either saline (total activity, A tau), 7 microM mipafox (AM1), or 0.8 mM mipafox (AM2): (BChE) = A tau-AM1, (AChE) = AM1-AM2, (Atypical ChE) = AM2. 3. The mipafox differential inhibition assay was used to determine the substrate hydrolysis patterns of myocardial cholinesterases with ASCh, acetyl-beta-methylthiocholine (A beta MSCh), propionylthiocholine (PSCh), and butyrylthiocholine (BSCh). The substrate specificities of myocardial AChE and BChE resemble those of erythrocyte AChE and serum BChE, respectively. Michaelis constants KM with ASCh were determined to be 0.15 mM for AChE and 1.4 mM for BChE. 4. Atypical cholinesterase, in respect to both substrate specificity and inhibition kinetics, differs from cholinesterase activities of vertebrate tissue and, up to now, could be identified exclusively in heart muscle. The enzyme's Michaelis constant with ASCh was determined to be 4.0 mM. 5. The reversible inhibitory effects of physostigmine (eserine) and quinidine on heart muscle cholinesterases were investigated using the differential inhibition assay. With all three isoenzymes, the inhibition kinetics of both substances were strictly competitive. The physostigmine inhibition of AChE was most pronounced (Ki = 0.22 microM). Quinidine most potently inhibited myocardial BChE (Ki = 35 microM).
Title: Cholinesterases of heart muscle. Characterization of multiple enzymes using kinetics of irreversible organophosphorus inhibition Chemnitius JM, Chemnitius GC, Haselmeyer KH, Kreuzer H, Zech R Ref: Biochemical Pharmacology, 43:823, 1992 : PubMed
Cholinesterases of porcine left ventricular heart muscle were characterized with respect to substrate specificity and inhibition kinetics with organophosphorus inhibitors N,N'-di-isopropyl-phosphorodiamidic fluoride (Mipafox), di-isopropylphosphorofluoridate (DFP), and diethyl p-nitro-phenyl phosphate (Paraoxon). Total myocardial choline ester hydrolysing activity (234 nmol/min/g wet wt with 1.5 mM acetylthiocholine, ASCh; 216 nmol/min/g with 30 mM butyrylthiocholine, BSCh) was irreversibly and covalently inhibited by a wide range of inhibitor concentrations and, using weighted least-squares non-linear curve fitting, residual activities as determined with four different substrates in each case were fitted to a sum of up to four exponential functions. Quality of curve fitting as assessed by the sum of squares reached its optimum on the basis of a three component model, thus, indicating the presence of three different enzymes taking part in choline ester hydrolysis. Final classification of heart muscle cholinesterases was obtained according to both substrate hydrolysis patterns with ASCh, BSCh, acetyl-beta-methylthiocholine and propionylthiocholine, and second-order rate constants for the reaction with organophosphorus inhibitors Mipafox, DFP, and Paraoxon. One choline ester-hydrolysing enzyme was identified as acetylcholinesterase (EC 3.1.1.7), and one as butyrylcholinesterase (EC 3.1.1.8). The third enzyme with relative resistance to organophosphorus inhibition was classified as atypical cholinesterase.
Title: Brain cholinesterases. Differentiation of target enzymes for toxic organophosphorus compounds Chemnitius JM, Haselmeyer KH, Zech R Ref: Biochemical Pharmacology, 32:1693, 1983 : PubMed
Cholinesterases in hen brain were characterized with respect to inhibition kinetics and substrate specificity. Three organophosphorus inhibitors were used: diethyl p-nitrophenyl phosphate (Paraoxon, E 600), di-isopropylphosphorofluoridate (DFP), and N,N'-di-isopropylphosphorodiamidic fluoride (Mipafox). The kinetics of irreversible cholinesterase inhibition were studied using two substrates, acetylthiocholine and butyrylthiocholine. The inhibition curves were analysed by the method of iterative elimination of exponential functions. Final classification of the different enzymes was done by combining two inhibitors in sequential inhibition expts. Six cholinesterases were shown to hydrolyse choline esters in hen brain, one was identified as acetylcholinesterase (EC 3.1.1.7) and one as cholinesterase (EC 3.1.1.8). Four enzymes can be classified as intermediate type cholinesterases according to their substrate specificity and to their inhibition constants. The possible role of different brain cholinesterases for the development of atypical symptoms following organophosphate intoxication is discussed.
The detoxication of organophosphorus compounds by phosphorylphosphatases was studied in primates. Taking into account the distribution of paraoxonase (EC 3.1.1.2) and DFPase (EC 3.8.2.1) in different tissues of the monkey (Macaca mulatta), the total detoxicating capacity for diethyl-p-nitrophenylphosphate (paraoxon, E 600) and diisopropylphosphorofluoridate (DFP) was determined. Acetylcholinesterase (AChE) (EC 3.1.1.7) of human brain was inhibited in vitro by paraoxon and DFP. Using the rate constants of AChE-inhibition and of AChE-synthesis those concentrations of organophosphorus inhibitors were calculated, which in vivo would reduce the steady-state AChE-activity to 20% of normal. This acute ineffective concentration is 7.6 X 10(-8) g/kg for DFP and 2.3 X 10(-8) g/kg for paraoxon. From substrate kinetics of the phosphorylphosphatases the time course of paraoxon and DFP detoxication in primates could be calculated. The time needed by phosphorylphosphatases to reduce a certain dose of an organophosphorus compound to the acute ineffective concentration is referred to as "effective detoxication time". The effective detoxication time (teff) was determined for different concentrations of paraoxon and DFP and was compared with the time needed by these organophosphate concentrations to inhibit AChE-activity to 12.5% of normal (t1/8). The significance of in vitro data for the evaluation of dose limits of organophosphate toxicity in vivo is discussed.
