Cholinesterases substrates

Scheme 6.2 Resolution of cholinesterase substrates from a thiolester DCL.

Larsson, R. Pei, Z. Ramstrom, O. Catalytic self-screening of cholinesterase substrates from a dynamic combinatorial thioester hbrary. Angew. Chem. Int. Ed. 2004,43, 3716-3718. 

Reiner, F., Simeon-Rudolf, V. (2000). Cholinesterase substrate inhibition and substrate activation. Pflugers Arch. Eur. J. Physiol. 440 R118-20. 

Cholinesterase Substrate Turnover number (minute ) Reference... 

B13. Beckett, A. H., Mitchard, M., and CUtherow, J. W., The importance of steric and stereochemical features in serum cholinesterase substrates. Biochem. Pharmacol. 17, 1601-1607 (1968). 

H17. Hastings, F. L., The study of horse and human serum cholinesterase substrate reactions. Ph.D. Thesis. Dept. Biochemistry, North Carolina State University, Ralei, 1966. 

The hydrolysis of cholinesterase substrates proceeds in three steps ... 

Skrinjarie-Spoljar, M., Frand.skovic, L., Radic, Z, Simeon, V and Reiner, E, (1992). Reaction of imidazolium and pyridinium oximes with the cholinesterase substrate acctylthiocholine. Acta P/wrm. 42, 77-83. 

A number of papers describe tedmiques for determination of cholinesterase activity based on amperometric measurement of products formed as a result of enzymatic hydrolysis (equation 1). In this case, artificial (butyryl or acetyl thiocholine) cholinesterase substrates are used. Thiocholine, formed as a result of cholinesterase-catalyzed hydrolysis can be measured amperometrically on a platinum electrode (14, 15) or mercury electrode (16). Analyses based on thiocholine determination employing an electrode modified by cobalt phthalocyanine (17-22) or cobalt tetraphenylporphyrin (23) have been described. Enzymatic hydrolysis of... 

Cholinesterases (ChEs), polymorphic carboxyles-terases of broad substrate specificity, terminate neurotransmission at cholinergic synapses and neuromuscular junctions (NMJs). Being sensitive to inhibition by organophosphate (OP) poisons, ChEs belong to the serine hydrolases (B type). ChEs share 65% amino acid sequence homology and have similar molecular forms and active centre structures [1]. Substrate and inhibitor specificities classify ChEs into two subtypes ... 

Similarly to quantitative determination of high surfactant concentrations, many alternative methods have been proposed for the quantitative determination of low surfactant concentrations. Tsuji et al. [270] developed a potentio-metric method for the microdetermination of anionic surfactants that was applied to the analysis of 5-100 ppm of sodium dodecyl sulfate and 1-10 ppm of sodium dodecyl ether (2.9 EO) sulfate. This method is based on the inhibitory effect of anionic surfactants on the enzyme system cholinesterase-butyryl-thiocholine iodide. A constant current is applied across two platinum plate electrodes immersed in a solution containing butyrylthiocholine and surfactant. Since cholinesterase produces enzymatic hydrolysis of the substrate, the decrease in the initial velocity of the hydrolysis caused by the surfactant corresponds to its concentration. Amounts up to 60 pg of alcohol sulfate can be spectrometrically determined with acridine orange by extraction of the ion pair with a mixture 3 1 (v/v) of benzene/methyl isobutyl ketone [271]. 

A number of substituted p-aminoacetates inhibit the enzyme cholinesterase. The main function of this enzyme is to hydrolyze acetyl choline and thereby terminate the action of that substrate as a neurotransmitter. Such inhibition is functionally equivalent to the administration of exogenous acetylcholine. Direct administration of the neurotransmitter substance itself is not a useful therapeutic procedure due to rapid drug destruction and unacceptable side... 

There are different types of cholinesterases in the human body, and they differ in their location in tissues, substrate affinity, and physiological function. The main ones are ACHE, present in nervous tissue and red blood cells (RBC-ACHE), and plasma cholinesterases (PCHE), present in glial cells, plasma, and liver. The physiological functions of RBC-ACHE and PCHE, if any, are unknown. 

Cholinesterases secreted by parasitic nematodes of (predominantly) the alimentary tract or other mucosal tissues are authentic AChEs when analysed by substrate specificity, inhibitor sensitivities and primary structure. In the first two respects, they resemble vertebrate AChEs, whereas somatic (and therefore presumably neuronal) enzymes of nematodes analysed to... 

Procedure Cholinesterase activity in analyzed tissue or the matrix (biotest with immobilized AChE) is determined in the incubation media [consisting of substrate ATCh - 34 mmol maleate buffer 0.1 M, pH = 6.0- 6.5 ml sodium citrate 0.1 M - 0.5 ml CuS045H20 0.03M -1.0 ml distilled H20 (or inhibitor in variant with toxin analyzed) -1.0 ml potassium ferricyanide 0.005 M -1 ml.] Volume of incubation media in one test - 400 mcl. As a blank (control sample), a treatment of the exposure without the substrate is used. If inhibitory effects of allelochemical (or any toxin) are analyzed, before the substrate addition the sample was preliminary exposed to allelochemical inhibitor. Two methods for the AChE-biotests may be recommended (i) in microcells ( stationary conditions ) and (ii) in flowing columns-reactors ( dynamic conditions ). 

Observations The preliminary treatment of the cholinesterase-containing material with allelochemical (or other compound, e.g. active oxygen species, ozone free radicals and peroxides, formed in allelopathic relations) is for 30 min, then a substrate acetylcholinesterase is added to the reaction medium and final reaction of hydrolysis is for 1 h. 

Organophosphate and carbamate pesticides are potent inhibitors of the enzyme cholinesterase. The inhibition of cholinesterase activity by the pesticide leads to the formation of stable covalent intermediates such as phosphoryl-enzyme complexes, which makes the hydrolysis of the substrate very slow. Both organophosphorus and carbamate pesticides can react with AChE in the same manner because the acetylation of the serine residue at the catalytic center is analogous to phosphorylation and carbamylation. Carbamated enzyme can restore its catalytic activity more rapidly than phosphorylated enzyme [17,42], Kok and Hasirci [43] reported that the total anti-cholinesterase activity of binary pesticide mixtures was lower than the sum of the individual inhibition values. 

In AChE-based biosensors acetylthiocholine is commonly used as a substrate. The thiocholine produced during the catalytic reaction can be monitored using spectromet-ric, amperometric [44] (Fig. 2.2) or potentiometric methods. The enzyme activity is indirectly proportional to the pesticide concentration. La Rosa et al. [45] used 4-ami-nophenyl acetate as the enzyme substrate for a cholinesterase sensor for pesticide determination. This system allowed the determination of esterase activities via oxidation of the enzymatic product 4-aminophenol rather than the typical thiocholine. Sulfonylureas are reversible inhibitors of acetolactate synthase (ALS). By taking advantage of this inhibition mechanism ALS has been entrapped in photo cured polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ) to prepare an amperometric biosensor for... 

Cholinesterases are widely distributed throughout the body in both neuronal and non-neuronal tissues. Based largely on substrate specificity, the cholinesterases are subdivided into the acetylcholinesterases (AChEs) (EC... 

Metabolism Substrate (minor) of Cholinesterase-medi- Substrate (minor) of... 

The contribution of pseudocholinesterase, also known simply as cholinesterase, to drug metabolism is much greater as it possesses considerably broader substrate selectivity. In addition to acetylcholine, it will hydrolyze other choline esters like the muscle relaxant succinylcholine. It will also hydrolyze non-choline-containing drugs like the local anesthetic procaine and the anti-inflammatory agent aspirin (Fig. 6.5). Cholinesterases, particularly... 

Fig. 12. Progress curve of inhibition of horse-serum cholinesterase by eserine and by di -isopropyl phosphorofluoridate in the absence of a substrate at pH 7-4 and 20°. x---x, 5x 10 8 M eserine O—O.ca. 3 x 10-10 M di-isopropyl phosphorofluoridate.

Effect of substrate concentration. In the following experiments the cholinesterase activities were measured by a continuous titration method. The digest of acetylcholine and horse-serum cholinesterase (total vol. 10 ml.), containing bromothymol blue and 0-0002 m phosphate, was titrated with 0-01 n NaOH to maintain the pH at 7-4. The titrations, which were carried out at 20°, were linear over a period of 10-15 min. The velocity was expressed as ml. 0-01 n NaOH/5 min. under the conditions used, it was proportional to the enzyme concentration. When an inhibitor was added, this was equilibrated with the enzyme, etc., for 5 min. at 20° before adding the substrate contained in a volume of 1 ml. 

Fig. 13. Effect of substrate concentration on inhibition of horse-serum cholinesterase.1 Enzyme activity was estimated by titration with 0-01 n NaOH at pH 7-4 and 20°. — , control, no inhibitor x — x, 2x 10 7 m eserine 0—O, 5 x 10 8 m di-isopropyl phosphorofluoridate.

True and pseudo-cholinesterase. The above serum preparations contained both the true and pseudo- cholinesterases of Mendel and Rudney.1 The effect of di-isopropyl phosphorofluoridate on these components was examined separately by means of the specific substrates described by Mendel, Mundel and Rudney,2 using the titration method described above. Phosphorofluoridate (5 x 10 8m) gave an inhibition of 57 per cent of the activity towards 00045m acetylcholine, 30 per cent of the activity towards 0-0005 m acetyl-/ methyl-choline, and 40 per cent of that towards 0-005 m benzoylcholine, after incubating the enzyme with the poison for 5 min. Thus in these experiments there appeared to be no appreciable difference in sensitivity of the true and pseudo-cholinesterases of horse serum to phosphorofluoridates. 

Benzoylcholine is a substrate of pseudo-cholinesterase but not of the true cholinesterase. It inhibits the true cholinesterase of man (laked red cells centrifuged and the supernatant liquid diluted 1/150, acetylcholine substrate 0 005 m) from 30 per cent at a concentration of 0-1 M to 85 per cent at a concentration of 0-3 M. Benzoylcholine injected intravenously into rabbits will, at a dose of 8-14 mg./kg. body weight, produce a head drop lasting 40-120 sec. 

Cholinesterases are subdivided into acetylcholinesterase and cholinesterase, one with a narrow, the other with broad substrate specificity [109-112], Both enzymes exist in multiple molecular forms distinguishable by their subunits association (Fig. 2.4). The hydrodynamic properties of these associations have allowed globular (G) and asymmetric (A) forms to be distinguished. The G forms can be hydrophilic (water-soluble, and excreted into body fluids) or amphiphilic (membrane-bound). The homomeric class exists... 

Semm albumin is not an enzyme but a transport protein, yet it has demonstrated hydrolytic activity against a variety of xenobiotic substrates. This este-rase-like activity has been known for years, but there is still confusion in the literature regarding its nature and mechanism. Indeed, it was not clear whether this activity is intrinsic to the albumin molecule or results from contamination of albumin preparations by one or more hydrolytic enzymes. More-recent studies with highly purified human serum albumin (HSA) have confirmed that the protein has an intrinsic esterase activity toward several substrates, but that activity due to contaminants and particularly semm cholinesterase is involved... 

Whereas the above evidence clearly points to a catalytic activity of serum albumin, it does not exclude an activity toward less-reactive substrates due to contamination of some HSA preparations. Indeed, the hypothesis of a contamination by plasma cholinesterase (EC 3.1.1.8) has been raised [126][127]. The efficient hydrolysis of nicotinate esters by HSA (see Chapt. 8) [128][129] could be due to contamination by cholinesterase in samples of a commercially available, essentially fatty acid free albumin. Support for this hypothesis was obtained when HSA contaminated with cholinesterase was resolved into two peaks by affinity chromatography, and the esterase activity toward nicotinate esters was found exclusively in the cholinesterase fraction [130],... 

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