The rate of acetylcholine hydrolysis of mammalian heart muscle influences cardiac responses to vagal innervation. We characterized cholinesterases of human left ventricular heart muscle with respect to both substrate specificity and irreversible inhibition kinetics with the organophosphorus inhibitor N,N'-di-isopropylphosphorodiamidic fluoride (mipafox). Specimens were obtained postmortem from three men and four women (61 +/- 5 years) with no history of cardiovascular disease. Myocardial choline ester hydrolyzing activity was determined with acetylthiocholine (ASCh; 1.25 mM), acetyl-beta-methylthiocholine (AbetaMSCh; 2.0 mM), and butyrylthiocholine (BSCh; 30 mM). After irreversible and covalent inhibition (60 min; 25 degrees C) with a wide range of mipafox concentrations (50 nM-5 mM), residual choline ester hydrolyzing activities were fitted to a sum of up to five exponentials using weighted least-squares non-linear curve fitting. In each ease, quality of curve fitting reached its optimum on the basis of a four component model. Final classification of heart muscle cholinesterases was achieved according to substrate hydrolysis patterns (nmol/min per g wet weight) and to second-order organophosphate inhibition rate constants k2 (1/mol per min); one choline ester hydrolyzing enzyme was identified as acetylcholinesterase (AChE; k2/mipafox = 6.1 (+/- 0.8) x 10(2)), and one as butyrylcholinesterase (BChE; k2/mipafox = 5.3 (+/- 1.1) x 10(3)). An enzyme exhibiting both ChE-like substrate specificity and relative resistance to mipafox inhibition (k2/mipafox = 5.2 (+/- 1.0) x 10(-1)) was classified as atypical cholinesterase.
Paraoxonase in serum and liver of rabbits and cattle was investigated. In serum the two substrates paraoxon and phenylacetate are exclusively hydrolyzed by alpha-lipoprotein-bound paraoxonase. In rabbit liver paraoxon is hydrolyzed only by paraoxonase, while phenylacetate is hydrolyzed by paraoxonase (20%) and additionally by an organophosphate sensitive carboxylesterase (B-Esterase), which is responsible for 80% of total liver phenylacetate hydrolysis. Phenyl acetate hydrolysis by B-Esterase of rabbit liver was shown to be inhibited by paraoxon and by mipafox covalently in a time and concentration dependent manner. Rabbit serum exhibits by far the highest serum paraoxonase activity (2.6 +/- 0.66 U/ml) of all vertebrate species tested up to now, while rabbit liver contains only 0.5 +/- 0.2 U/g fresh weight. In cattle extremely high paraoxonase activity is found in liver (2.8 U/g), while bovine serum contains only 0.2 U/g. The paraoxonase activity ratio (hydrolysis rate paraoxon: phenylacetate x 1000) in cattle does not show interindividual variation (activity ratio 4.0 +/- 0.4, correlation coefficient 0.996, P < 0.001). In contrast, the paraoxon/phenylacetate hydrolysis ratio of rabbit paraoxonase in serum as well as in liver does vary considerably between individuals. In cross-bred rabbits paraoxonase activity ratios from three to ten are found. In a strain of pure-bred New Zealand White rabbits three polymorphic serum paraoxonase phenotypes could be clearly differentiated by the activity ratio. By analogy with the human paraoxonase polymorphism, the rabbit paraoxonase isotypes were classified as paraoxonase A (activity ratio 3.8-4.3), AB (ratio 5.5-6.0) and B (ratio 7.3-8.6). The corresponding frequencies of the three isotypes were 40, 35 and 25%.
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: Computerized analysis of covalent inhibition kinetics for identification of heart muscle cholinesterase and brain carboxylesterase isoenzymes. Design of differential inhibition assays Chemnitius JM, Dewald K, Kreuzer H, Zech R Ref: Chemico-Biological Interactions, 87:239, 1993 : PubMed
The kinetics of time- and concentration-dependent covalent organophosphorus inhibition of carboxylesterase isoenzymes (EC 3.1.1.1) and cholinesterase isoenzymes (EC 3.1.1.7 and EC 3.1.1.8) were investigated using a wide range of organophosphate inhibitor concentrations (10(-10)-10(-3) mol/l) and different inhibition times. Computerized analysis of inhibition curves by weighted non-linear least-squares curve fitting was compared to graphic analysis by iterative elimination of exponential functions. Possible experimental errors due to inhibitor saturation kinetics and enzymatic organophosphate hydrolysis were thoroughly investigated. In mammalian heart muscle, three different cholinesterase isoenzymes were identified. High sensitivity and specificity of the classic differential inhibition test for carboxylesterase activity of hen brain neuropathy target esterase (NTE) could be confirmed independently with both methods of inhibition curve analysis.
Paraoxonase of human and animal sera was shown to be a structural part of high density lipoproteins (HDL) by immunoprecipitation, heparin- or polyethyleneglycol fractionation, ultracentrifugation and gel chromatography. Frequency distribution of paraoxonase activity in human sera is trimodal. Human individuals, with respect to paraoxon detoxication, can be distinguished into low and high detoxicators using ratios of phenylacetate and paraoxon hydrolysis as well as activation with ethanolamine and sodium chloride. With conversion of alpha-lipoprotein subtype HDL3 to HDL2, specific activities of paraoxonase and arylesterase are increasing about 3.5-fold in low detoxicator individuals and 1.9-fold in high detoxicators, indicating that more than 90% of HDL2 particle-bound paraoxonase and arylesterase activity are incorporated during the HDL conversion process. HDL cholesterol concentrations in individual sera were shown to be positively correlated to both serum paraoxonase and arylesterase activities.
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.
Title: Carboxylesterases in primate brain: characterization of multiple forms Chemnitius JM, Zech R Ref: International Journal of Biochemistry, 15:1019, 1983 : PubMed
Carboxylesterase activity of primate brain (Macaca mulatta) was determined using phenyl valerate (PV) as substrate. Eight carboxylesterases of primate brain were characterized in respect to PV-hydrolysing activity and to their inhibition rate constants for the reaction with organophosphorus compounds. Carboxylesterase III was identified as neurotoxic esterase (NTE). Organophosphate inhibition data of primate acetylcholinesterase (EC 3.1.1.7) and of primate cholinesterase (EC 3.1.1.8) were determined and are compared to corresponding data of primate brain carboxylesterases. Physiological functions, clinical and toxicological significance of primate brain carboxylesterases are 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.
Title: Inhibition of acetylcholinesterase by anisomycin Zech R, Domagk GF Ref: Life Sciences, 19:157, 1976 : PubMed
Title: [A micromethod for the simultaneous determination of acetylcholinesterase and butyrylcholinesterase in mammalian blood] Zech R, Franke K, Domagk GF Ref: Z Klin Chem Klin Biochem, 7:547, 1970 : PubMed
Title: Grenzen der Therapie mit Oximen bei Vergiftungen mit insekticiden Alkylphosphaten Zech R, Erdmann WD, Engelhard N Ref: Arzneimittelforschung, 17:1196, 1967 : PubMed
Title: Zur Frage der therapeutischen Wirksamkeit von Esterase-Reaktivatoren bei der Vergiftung mit Dimethoat Erdmann WD, Zech R, Franke P, Bosse I Ref: Arzneimittelforschung, 16:492, 1966 : PubMed
Title: Reaktionen von Pyridinium Oximen mit dem Alkylphosphat Dimethoat und seinen Derivaten. Wirkung auf Cholinesterasen Zech R, Engelhard H, Erdmann W-D Ref: Biochimica & Biophysica Acta, 128:363, 1966 : PubMed
