Pseudomonas aeruginosa cholinesterase and phosphorylcholine phosphatase: two enzymes contributing to corneal infection

Choline, acetylcholine and betaine used as the sole carbon, nitrogen or carbon and nitrogen source increase cholinesterase activity in addition to phosphorylcholine phosphatase and phospholipase C activities in Pseudomonas aeruginosa. The cholinesterase activity catalyses the hydrolysis of acetylthiocholine (Km approx. 0.13 mM) and propionylthiocholine (Km approx. 0.26 mM), but not butyrylthiocholine, which is a pure competitive inhibitor (Ki 0.05 mM). Increasing choline concentrations in the assay mixture decreased the affinity of cholinesterase for acetylthiocholine, but in all cases prevented inhibition raised by high substrate concentrations. Considering the properties of these enzymes, and the fact that in the corneal epithelium there exists a high acetylcholine concentration and that Pseudomonas aeruginosa produces corneal infection, it is proposed that these enzymes acting coordinately might contribute to the breakdown of the corneal epithelial membrane.     

Comparative study of enzymatic activity of cholinesterases of different origin in thionaphthylacetate hydrolysis reactions

A comparative determination of kinetic parameters V and Km in the reaction of hydrolysis thionaphthylacetate and well known substrate acetylthiocholine by choline esterases from different sources was conducted. It is shown that butyrylcholine esterases hydrolyze thionaphthylacetate with velocity comparable with that of hydrolysis of acetylthiocholine, while acetylcholine esterases and propionylcholine esterases hydrolyze this substrate several times slower than acetylthiocholine. The values of Km in the reactions of hydrolysis of thionaphthylacetate for all studied cholinesterases is an order higher than for acetylthiocholine except cholinesterase of blood serum of fish. This value for the latter enzyme is practically equal.     

Use of 1- and 2-thionaphthylacetates as cholinesterase substrates

1- and 2-thionaphthylacetates were tested as cholinesterase substrates. It was shown that the butyrilcholinesterase from horse serum can hydrolize these compounds. The hydrolysis velocity of 1-thionaphthylacetate was comparable with hydrolysis velocity of acetylthiocholine (the well known cholinesterase substrate), but 2-thionaphthylacetate was hydrolysed more slowly. The values of the kinetic parameters V and K(m) for butyrylcholinesterase hydrolysis of 1- and 2-thionaphthylacetates were determined. It was offered to use 1-thionaphthylacetates as the substrate for cholinesterases.     

Complete Staining of Nerve Fiber and Myoneural Junctions with Acetylthiocholine and Silver

We describe a combined stain for simultaneous demonstration of the preterminal axons and cholinesterase activity at myoneural junctions of mammalian muscles. This technique employs acetylthiocholine iodide as the substrate for cholinesterase activity and silver nitrate impregnation of preterminal axons. The procedure is rapid, simple and Uses fresh muscles. Intramuscular nerves, preterminal axons and myoneural junctions are stained simultaneously brown or black with minimal background staining of connective tissue and muscle fibers.     

Complete Staining of Nerve Fiber and Myoneural Junctions with Acetylthiocholine and Silver

We describe a combined stain for simultaneous demonstration of the preterminal axons and cholinesterase activity at myoneural junctions of mammalian muscles. This technique employs acetylthiocholine iodide as the substrate for cholinesterase activity and silver nitrate impregnation of preterminal axons. The procedure is rapid, simple and Uses fresh muscles. Intramuscular nerves, preterminal axons and myoneural junctions are stained simultaneously brown or black with minimal background staining of connective tissue and muscle fibers.     

Cholinesterases from Plant Tissues: II. Inhibition of Bean Cholinesterase by 2-Isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine Carboxylate Methyl Chloride (AMO-1618)

A cholinesterase was purified 36-fold from mung bean (Phaseolus aureus) roots by a combination of differential extraction media and gel filtration. The enzyme could be effectively extracted only by high salt concentration, indicating that it is probably membrane-bound. Methods used for assaying animal cholinesterases were tested, two of which were adapted for use with the bean cholinesterase. The bean enzyme hydrolyzed choline and noncholine esters but showed its highest affinity for acetylcholine and acetylthiocholine. The pH optimum was 8.5 for acetylthiocholine and 8.7 for acetylcholine. The Michaelis constants were 72 and 84 μm for acetylcholine and acetylthiocholine, respectively. The cholinesterase was relatively insensitive to eserine (half-maximum inhibition at 0.42 mm) but showed high sensitivity to neostigmine (half-maximum inhibition at 0.6 μm). Other animal cholinesterase inhibitors were also found to inhibit the bean enzyme but most of them at higher concentrations than are generally encountered. Choline stimulated enzymatic activity. The molecular weight of the cholinesterase was estimated to be greater than 200,000, but at least one smaller form was observed. It is suggested that the large form of cholinesterase is converted to the smaller form by proteolysis.     

Cholinesterases from plant tissues: I. Purification and characterization of a cholinesterase from mung bean roots

A cholinesterase was purified 36-fold from mung bean (Phaseolus aureus) roots by a combination of differential extraction media and gel filtration. The enzyme could be effectively extracted only by high salt concentration, indicating that it is probably membrane-bound. Methods used for assaying animal cholinesterases were tested, two of which were adapted for use with the bean cholinesterase. The bean enzyme hydrolyzed choline and noncholine esters but showed its highest affinity for acetylcholine and acetylthiocholine. The pH optimum was 8.5 for acetylthiocholine and 8.7 for acetylcholine. The Michaelis constants were 72 and 84 mum for acetylcholine and acetylthiocholine, respectively. The cholinesterase was relatively insensitive to eserine (half-maximum inhibition at 0.42 mm) but showed high sensitivity to neostigmine (half-maximum inhibition at 0.6 mum). Other animal cholinesterase inhibitors were also found to inhibit the bean enzyme but most of them at higher concentrations than are generally encountered. Choline stimulated enzymatic activity. The molecular weight of the cholinesterase was estimated to be greater than 200,000, but at least one smaller form was observed. It is suggested that the large form of cholinesterase is converted to the smaller form by proteolysis.     

Hemolymph cholinesterase activity in the Mussel <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> (Dunker, 1853) that was exposed to adverse natural and anthropogenic conditions

The influence of habitat conditions on the activity, the structure of the substrate specificity (the ratio of the substrate hydrolysis rates), and the kinetic parameters of substrate hydrolysis due to the effect of hemolymph cholinesterase of the mussel Crenomytilus grayanus was studied. Mussels were collected from areas that are influenced by seasonal and stationary upwelling, as well as from a polluted area. Upwelling and anthropogenic pressure were shown to alter the structure of hemolymph cholinesterase substrate specificity in mussels, up to complete loss of the ability to catalyze the hydrolysis of propionyland butyrylthiocholine. It was established that during the seasonal upwelling the efficiency of the cholinergic process in mussels is provided by a wide range of effective concentrations of the substrates and by decreasing their affinity to the enzyme. Under the conditions of chronic anthropogenic pollution, the cholinesterase of the mussel hemolymph loses its ability to hydrolyze substrates other than acetylthiocholine.     

Effect of salt composition of the medium and ethanol on cholinesterase hydrolysis of various substrates

Sodium chloride, phosphate buffer and ethanol were studied for their effect on butyryl cholinesterase hydrolysis rate of acetylcholine, acetylthiocholine, butyrylthiocholine and nonion substrate of indophenylacetate. The concentrations of 1.10(-2) = 1.10(-1) M of sodium chloride activated enzymatic hydrolysis of ion substrates at the concentrations lower than 1.10(-4) M but sodium chloride is a competitive inhibitor at higher concentrations. Phosphate buffer also activates substrates enzyme hydrolysis at the concentrations of 2.10(-4) M and lower, but it inhibits incompetitively the nonion substrate indophenylacetate hydrolysis. Ethanol activates butyrylthiocholine hydrolysis and is a competitive inhibitor in acetylthiocholine and indophenylacetate hydrolysis. The observed effects are discussed on the assumption of two forms of butyrylcholinesterase E' and E" existence. These two forms are determined by different kinetic parameters and are in equilibrium.     

Cholinesterases from flounder muscle. Purification and characterization of glycosyl-phosphatidylinositol-anchored and collagen-tailed forms differing in substrate specificity

Flounder (Platichthys flesus) muscle contains two types of cholinesterases, that differ in molecular form and in substrate specificity. Both enzymes were purified by affinity chromatography. About 8% of cholinesterase activity could be attributed to collagen-tailed asymmetric acetylcholinesterase sedimenting at 17S, 13S and 9S, which showed catalytic properties of a true acetylcholinesterase. 92% of cholinesterase activity corresponded to an amphiphilic dimeric enzyme sedimenting at 6S in the presence of Triton X-100. Treatment with phospholipase C yielded a hydrophilic form and uncovered an epitope called the cross-reacting determinant, which is found in the hydrophilic form of a number of glycosyl-phosphatidylinositol-anchored proteins. This enzyme showed catalytic properties intermediate to those of acetylcholinesterase and butyrylcholinesterase. It hydrolyzed acetylthiocholine, propionylthiocholine, butyrylthiocholine and benzoylthiocholine. The Km and the maximal velocity decreased with the length and hydrophobicity of the acyl chain. At high substrate concentrations the enzyme was inhibited. The p(IC50) values for BW284C51 and ethopropazine were between those found for acetylcholinesterase and butylcholinesterase. For purified detergent-soluble cholinesterase a specific activity of 8000 IU/mg protein, a turnover number of 2.8 x 10(7) h-1, and 1 active site/subunit were determined.     

The cholinesterase properties of Daphnia magna

It has been demonstrated that cholinesterase of Daphnia magna is capable of the hydrolysis of propionylthiocholine iodide at the highest rate as compared to the other substrates studied, the hydrolysis being inhibited by high concentrations of the substrate. The rate of splitting of acetylthiocholine iodide is similar to that of propionylthiocholine iodide, whereas the hydrolysis of butyrylthiocholine iodide is 3 times slower. Cholinesterase from D. magna is extremely sensitive to an organophosphorus inhibitor, DDVP. The value of bimolecular constant of the inhibition rate (kappa II) is equal to (1.60 +/- 0.20).10(8)1.mol-1.min-1.     

Characterization of cholinesterase of muscularis muscle of Bufo marinus

We have characterized the cholinesterase (ChE) of muscularis muscle of Bufo marinus by selectively using specific inhibitors of acetylcholinesterase and pseudocholinesterase and observing susceptibility to inhibition when substrate is present in excess. The ChE activity in this preparation due to acetylcholinesterase (AChE) and pseudocholinesterase (BuChE) was 90 and 10%, respectively. The optimum temperature and pH for the ChE were 38 degrees C and 7.4, respectively and the excess substrate inhibition was noted above a pS of 2.6. The Km for acetylthiocholine (ASCh) was 0.76 X 10(-4) M.     

An enzyme from the fungus Aspergillus niger that hydrolyzes choline ethers]

The acetylthiocholine-hydrolyzing enzymatic activity inhibited by the neostigmine and partly physostigmine has been found in extracts from mycelium of fungus Aspergillus niger. The enzyme has been isolated and 15-20 fold purified. The cholinesterase activity of the protein (Kmu 7.10-7 M) is comparable with known for analogous enzymes from higher plants, for its inhibition high concentrations of substrate (greater than 10-3M) are required. The enzyme hydrolyzes acetylthiocholine with rate approximately 1.5 times higher than butyrylthiocholine. Molecular mass of native protein is approximately 600 kDa, subunits -63 and 44 kDa.     

Postnatal changes in the activities of acetylcholinesterase and butyrylcholinesterase in rat heart atria

Postnatal development of the activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in rat heart atria has been investigated with the use of 1.5-bis (allyldimethylammoniumphenyl) pentane-3-dibromide (BW 284 C51) as a selective inhibitor of AChE. Total cholinesterase activity (mumol acetylthiocholine hydrolysed X g-1 per hour) increased from 218 on the 1st day after birth to 426 on the 30th day and diminished to 340 in adult rats. The activity of AChE (mumol acetylthiocholine hydrolysed X g-1 per hour) underwent more dramatic changes, increasing more than 4-fold during the first month of life, from 13 on the 1st to 58 on the 30th day of life and then decreasing to 42 in adult rats. The proportion of AChE on total cholinesterase activity increased from 6% on the 1st day to 12-15% in animals aged 24 days and more. Since AChE is known to be specifically involved in the termination of the action of acetylcholine in the sinoatrial node, the observed postnatal changes in its activity are likely to play a role in the postnatal development of cardiac parasympathetic control.     

Effect of pH on the activity of nervous cholinesterases of the rat towards different biochemical and histochemical substrates and inhibitors

Summary Cholinesterase activities of homogenates of rat brain and superior cervical ganglion were determined by automatic titration using several biochemical and histochemical substrates. High hydrolysis rates were observed when acetylcholine, acetylthiocholine, propionylcholine or propionylthiocholine was used as substrate; -naphthyl acetate and acetyl--methylcholine were hydrolyzed at a moderate rate, and activities were low towards butyrylcholine, butyrylthiocholine and benzoylcholine. With most substrates, the enzyme activity increased from pH 5 to pH 10 and decreased at pH 11. Acetylcholine and acetyl--methylcholine showed an activity maximum at pH 7 or 8. Inhibition by the selective inhibitor of specific cholinesterase 284 C 51 was not markedly affected by pH. On the other hand, the inhibiting power of the selective inhibitor of non-specific cholinesterase iso-OMPA markedly decreased when the pH was lowered. The inhibitor data at different pHs and with different concentrations of eserine, 284 C 51 or iso-OMPA at pH 6 indicated that acetylcholine, propionylcholine and propionylthiocholine are readily hydrolyzed by both specific and non-specific cholinesterase, while acetyl--methylcholine is mainly split by specific cholinesterase and butyrylcholine mainly by non-specific cholinesterase. The significance of propionylcholine and propionylthiocholine as substrates of specific cholinesterase is emphasized.     

Localization of cholinesterase in Pseudomonas aeruginosa strain K

The inducible cholinesterase of Pseudomonas aeruginosa strain K (ATCC 25102) degraded propionylcholine, acetylthiocholine, acetylcholine and acetyl-beta-methylcholine at a high rate and butyrylcholine and succinylcholine at very low rates. The localization of the enzyme in the periplasmic space was indicated by a similar rate of acetylcholine degradation by intact cells or their extracts, by release of cholinesterase together with alkaline phosphatase into the culture medium during cell growth in a low phosphate-containing medium, by liberation of cholinesterase and alkaline phosphatase during lysozyme-induced conversion of cells to spheroplasts and by freezing and thawing. Threatment of cells with diazo-7-amino-1,3-naphthalenedisulphonic acid, which inactivates surface-located enzymes, abolished most of the cholinesterase and 5'-nucleotidase activities.     

Cholinesterases from plant tissues. VI. Preliminary characterization of enzymes from Solanum melongena L. and Zea mays L.

Enzymes capable of hydrolyzing esters of thiocholine have been assayed in extracts of Solanum melongena L. (eggplant) and Zea Mays L. (corn). The enzymes from both species are inhibited by the anti-cholinesterases neostigmine, physostigmine, and 284c51 and by AMO-1618, a plant growth retardant and they both have pH optima near pH 8.0. The enzyme from eggplant is maximally active at a substrate concentration of 0.15 mM acetylthiocholine and is inhibited at higher substrate concentrations. On the basis of this last property, the magnitude of inhibition by the various inhibitors, and the substrate specificity, we conclude that the enzyme from eggplant, but not that from corn, is a cholinesterase.     

Oxidation of cholinesterase-inhibiting pesticides: A simple experiment to illustrate the role of bioactivation in the toxicity of chemicals

A simple undergraduate laboratory experiment that can be used in Biochemistry and Toxicology courses to illustrate the importance of metabolic reactions in the toxicity of chemical substances is reported. It involves the experimental confirmation that oxidized phosphorothionate esters, commonly used as insecticides, are stronger cholinesterase inhibitors and therefore exhibit higher toxicity than do their sulphur analogs starting from which the first are formed by in vivo oxidative desulphuration. Two separated aliquots of a bovine blood sample are incubated with parathion and paraoxon, its oxygen analog, and compared for cholinesterase activity with "normal" blood. Previously, a standard sample of paraoxon was obtained by oxidation of the thiono group of parathion with bromine vapour by reaction TLC. The comparison of the inhibitory capacity of both compounds is made by a colorimetric procedure using acetylthiocholine as substrate of the enzyme and 5,5'-dithiobis-(2-nitrobenzoic acid) as chromogen.     

Microscopic localization of cholinesterases in the nervous systems of the lobsters, Panulirus argus and Homarus americanus

Using acetylthiocholine as substrate, microscopically localizable cholinesterase (ChE) activity is demonstrated in neural and glial elements of central and peripheral nervous systems of the lobsters, Panulirus argus and Homarus americanus. Moderate to very intense ChE activity occurs in all synaptic regions of the central ganglia and stomatogastric ganglion, in glial sheaths around neuron somata and peripheral nerve axons, and in cytoplasm of a few nerve cell bodies. Axons, identified as motor, contain extremely little ChE. The principal reaction in peripheral nerves occurs in sheath elements of sensory fibres; in most cases, much of the reaction is lost as the nerves lose the sheaths at the point of entry into brain.     

Mutant of Bungarus fasciatus acetylcholinesterase with low affinity and low hydrolase activity toward organophosphorus esters

Enzymes hydrolysing highly toxic organophosphate esters (OPs) are promising alternatives to pharmacological countermeasures against OPs poisoning. Bungarus fasciatus acetylcholinesterase (BfAChE) was engineered to acquire organophosphate hydrolase (OPase) activity by reproducing the features of the human butyrylcholinesterase G117H mutant, the first mutant designed to hydrolyse OPs. The modification consisted of a triple mutation on the (122)GFYS(125) peptide segment, resulting in (122)HFQT(125). This substitution introduced a nucleophilic histidine above the oxyanion hole, and made space in that region. The mutant did not show inhibition by excess acetylthiocholine up to 80 mM. The k(cat)/K(m) ratio with acetylthiocholine was 4 orders of magnitude lower than that of wild-type AChE. Interestingly, due to low affinity, the G122H/Y124Q/S125T mutant was resistant to sub-millimolar concentrations of OPs. Moreover, it had hydrolysing activity with paraoxon, echothiophate, and diisopropyl phosphofluoridate (DFP). DFP was characterised as a slow-binding substrate. This mutant is the first mutant of AChE capable of hydrolysing organophosphates. However, the overall OPase efficiency was greatly decreased compared to G117H butyrylcholinesterase.