Ronald Zech

Organophosphate detoxicating hydrolases in different vertebrate species.

Phosphorylphosphatase activities in various organs of vertebrate species from different classes were determined using a spectrophotometric assay for paraoxonase (EC 3.1.1.2) and a potentiometric assay with a fluoride sensitive electrode for DFPase (EC 3.8.2.1). Temperature-dependent inactivation experiments, an extended interpretation of mixed substrate studies and activity distribution patterns confirm that in vertebrate tissue at least two different enzymes are responsible for hydrolytic detoxication of paraoxon and DFP. Total organophosphate detoxicating phosphorylphosphatase activity of a certain animal species is shown to be the major determinant for differences between the inhibitory potency of organophosphorus compounds on the animals target enzymes in vitro and organophosphate toxicity in vivo.

Determination of peroxides in saliva-kinetics of peroxide release into saliva during home-bleaching with Whitestrips® and Vivastyle®

Aim of the study was to determine peroxides in saliva, released during bleaching procedures. Upper incisors of five subjects were bleached with Whitestrips (5% H2O2) and Vivastyle (10% carbamide peroxide, tray charged with 225mg) for 30min, each on different days. Saliva was collected before and during the whole period of bleaching at different intervals. The amount of peroxide in the salivary samples was assessed with peroxidase, phenol and 4-aminoantipyrin in a photometric assay. Additionally the amount of peroxides in the bleaching material was determined before and after the bleaching, so that the peroxide release into saliva could be balanced. The amount of peroxides released into saliva was related to the bleaching system and only partially influenced by the individual salivary flow rate. Bleaching with Vivastyle led to lower release of peroxides into saliva compared to Whitestrips (Vivastyle: 0.8+/-0.17mg; Whitestrips: 1.5+/-0.84mg). Salivary flow rate was not correlated to release of peroxides from the bleaching products. It can be concluded that the enzymatic method adopting 4-aminoantipyrin and peroxidase is valid for the determination of peroxides in saliva. Furthermore distinctly more peroxides are released into the oral cavity from Whitestrips than from trays charged with Vivastyle .

Rapid preparation of subsarcolemmal and interfibrillar mitochondrial subpopulations from cardiac muscle

1. Subsarcolemmal and interfibrillar mitochondria were prepared with complete recovery from rabbit and porcine heart muscle by upward-flotation during 60 sec of Percoll density gradient centrifugation. 2. Mitochondrial subpopulations were identified and characterized according to buoyant density, electron-microscopy, marker enzyme activities and respiratory performance. 3. ADP-induced state 3-respiration related to latent citrate synthase activity as a marker for structurally intact mitochondria was not significantly different in both mitochondrial subtypes.

Lipoproteins and hydrolysis of organophosphorus compounds

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.

Neurotoxic esterase: Gel filtration and isoelectric focusing of carboxylesterases solubilized from hen brain

Carboxylesterase activity (EC 3.1.1.1) of hen brain including neurotoxic esterases NTEA and NTEB is solubilized from lyophilized lipid-extracted brain material by the use of n-octylglucoside. The solubilized enzymes are subjected to free isoelectric focusing, six carboxyl - esterase activity peaks are obtained. By gel filtration on Sephacryl S-300 neurotoxic esterases are separated from carboxylesterase isoenzymes V and X. The molecular weight of the neurotoxic esterases is estimated to be 1.8 X 10(6).

Identification of isoenzymes in cholinesterase preparations using kinetic data of organophosphate inhibition

Abstract Commercial preparations of acetylcholinesterase (EC 3.1.1.7) and of cholinesterase (EC 3.1.1.8) were characterized by organophosphate inhibition. Cholinesterase activities were inhibited by varying organophosphate concentration and time of inhibition. Bimolecular rate constants were determined by plotting log activity vs inhibitor concentration or inhibition time. Inhibition of acetylcholinesterase from bovine erythrocytes by diethyl p-nitrophenyl phosphate (Paraoxon), diisopropylphosphorofluoridate (DFP), and N,N′-diisopropylphosphorodiamidic fluoride (Mipafox) in semilogarithmic plots showed a linear decay of activity. Inhibition of acetylcholinesterase from electric eel (Electrophorus electricus) and of cholinesterases from horse serum and from human serum did not show linear characteristics, indicating the presence of more than one single enzyme in these preparations. The corresponding inhibition curves were resolved by subtraction of exponential functions. In each case two different activity components were identified and characterized in respect to partial activity, substrate specificity, and reactivity with organophosphorous compounds. The suitability of the method for application on crude homogenates is discussed.

Inhibition of acetylcholinesterase by anisomycin

Abstract The antibiotic anisomycin, an inhibitor of protein synthesis in eucaryotic cells, which blocks long-term memory in mice, is shown to interact with the cholinergic system by inhibiting reversibly the acetylcholinesterase. The inhibition is a competitive one, the inhibition constant K i being 5.0 × 10 −3 for human brain acetylcholinesterase and 1.7 × 10 −3 for acetylcholinesterase of bovine erythrocytes. The anisomycin effect on acetylcholinesterase is compared with the puromycin and cycloheximide-inhibition of the enzyme. The significance of the cholinergic effect of anisomycin in addition to its inhibitory effect on protein synthesis for the interpretation of memory experiments is discussed.

Carboxylesterases in primate brain: characterization of multiple forms

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.

Paraoxonase polymorphism in rabbits

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%.

Preparation of two neurotoxic esterases from the chick central nervous system

A standard procedure for lipid-extraction of lyophilized hen brain material is described. Nine carboxylesterase isoenzymes (EC 3.1.1.1) are identified in lipid-extracted lyophilized material (LELM) using kinetic analysis of organophosphate inhibition. Total phenyl valerate (PV) hydrolysing carboxylesterase activity in LELM is 43.3 U X g-1. Two carboxylesterase isoenzymes of LELM are classified as neurotoxic esterases (NTEA and NTEB). Using n-octylglucoside 51% of the water-insoluble neurotoxic esterase activity from LELM are solubilized. Six carboxylesterase isoenzymes including NTEA (6.5 U X 1(-1] and NTEB (4.2 U X 1(-1] are present in the solubilized preparation. Throughout purification and separation steps carboxylesterase isoenzymes are identified by their rate constants for the reaction with organophosphorus inhibitors.