Cholinesterase surfaces

The cholinesterase surfaces described here, as in the case of OPH, are broad spectrum. The TAC and TBC surfaces indicate only the presence of a competitive inhibitor of the enzyme and not its identity, so a positive result would be observed upon exposure to an organophosphate or carbamate pesticide, nerve agent, or drug with anti-cholinesterase activity. When the two enzymes are combined on a single surface, classes of compounds can be distinguished, that is, the surface discriminates between competitive inhibitors of AChE only, competitive inhibitors of BChE only, and competitive inhibitors of AChE and BChE. Inhibitor identification can be made by comparison of the change in absorbance at the AChE peak as compared to that for the BChE peak on the combined surface. 

The aim of our investigation was the development of the amperometric enzyme immunosensor for the determination of Klebsiella pneumoniae bacterial antigen (Ag), causes the different inflammatory diseases. The biosensing pail of the sensors consisted of the enzyme (cholinesterase) and antibodies (Ab) immobilized on the working surface of the screen-printed electrode. Bovine seiaim albumin was used as a matrix component. 

The Structure of the Active Surface of Cholinesterases and the Mechanism of Their Catalytic Action in lister Hydrolysis... 

Fig. 11.2. Schematic representation of the primary structure of secreted AChE B of N. brasiliensis in comparison with that of Torpedo californica, for which the three-dimensional structure has been resolved. The residues in the catalytic triad (Ser-His-Glu) are depicted with an asterisk, and the position of cysteine residues and the predicted intramolecular disulphide bonding pattern common to cholinesterases is indicated. An insertion of 17 amino acids relative to the Torpedo sequence, which would predict a novel loop at the molecular surface, is marked with a black box. The 14 aromatic residues lining the active-site gorge of the Torpedo enzyme are illustrated. Identical residues in the nematode enzyme are indicated in plain text, conservative substitutions are boxed, and non-conservative substitutions are circled. The amino acid sequence of AChE C is 90% identical to AChE B, and differs only in the features illustrated in that Thr-70 is substituted by Ser.

Experiment 2. Cholinesterase as a sensor on the cell surface A target of the allelochemical may also be a surface sensor-cholinesterase (Fig. 10). If after the staining with Red analogue of Ellman reagent the blue colour is absent in the allelochemical treated microspore, possible target is the enzyme (Roshchina, 2001a,b) as for alkaloid berberine tested. If after the treatment by the test allelochemical, the colour is absent or light, the compound inhibits the enzyme (also see biochemical assay in Chapter 11). 

Blue colour ring concentrated on plasmalemma of pollen (A). Arrows on B (lower part) shows a difference in the colour between cellular surface (blue color) and apperture for the output of pollen tube (red colour). Pistil excreted blue colour product, which covers the red coloured surface of pistil. The cholinesterase activity in plants is considered as sensitive test to study the allelopathic activity (Roshchina and Roshchina 1993 Roshchina,1999 2001a). 

May cause severe and painful irritation of the eyes, nose, throat, and lungs. Severe exposure can cause accumulation of fluid in the lungs (pulmonary edema). Inhalation toxicity similar to hydrogen chloride and hydrogen fluoride. May cause second or third degree burns upon short contact with skin surfaces. Oral ingestion may result in tissue destruction of the gastrointestinal tract. Decreased blood cholinesterase levels have been reported in animals. 

The primary and tertiary structures of the cholinesterases are known. The primary structures of the cholinesterases initially defined a large and functionally eclectic superfamily of proteins, the a,P hydrolase fold family, that function not only catalytically as hydrolases but also as surface adhesion molecules forming heterologous cell contacts, as seen in the structurally related proteins... 

The ester must in addition contain some group which will initiate the approach of the ester to the surface of the enzyme. In this connexion it should be noted that di-isopropyl phos-phorochloridate (III, X = Cl), in which the chlorine atom is chemically very reactive,3 has no toxic properties, is devoid of myotic and anti-cholinesterase activity. In this compound, the chlorine is hydrolysed very quickly in water and would probably be destroyed extremely quickly in vivo. We have shown, quite conclusively, that in non-polar solvents the phosphorochloridate... 

Chloropentammine Ir (HI) complex, incomplete Ir (III) autoreduction, 39 151-152 Chloroplasts, quantum conversion in, 14 1 1 -Chloroprop-2-ene thermal decomposition, 41 80 Chlorpromazine, reactivity with EDA complexes, 20 333, 336 CH O, 32 374-375 CH3OH, oxidation, 38 21-23 Cholestenone, hydrogenation, 25 57, 58 Cholesterol, biosynthesis of, 25 382 Cholinesterases, stracture of active surface, 10 130... 

Most cholinesterase inhibitors inhibit the enz)nne by acylating the esteratic site on the enzyme surface. Physostigmine and neostigmine are examples of... 

Fleisher reported that both sarin and VX increased the sensitivity of the isolated frog s rectus abdominis to external application of acetylcholine and at the same time decreased the activity of cholinesterase in the external surfaces of the muscle cells. Sarin at 5 x 10-7 M reduced the threshold concentration of acetylcholine for inducing contraction of the muscle to 8% of that required before application of sarin. The same concentration of VX reduced the threshold concentration of acetylcholine to 6.7% of that needed previously. Contemporaneously, the activity of cholinesterase in the external surfaces of the muscle cells was reduced to 8.1% and 0% of that before application of sarin and VX, respectively. Addition of 2-PAM I at 5 x 10 M to the baths in which the muscles were suspended had little effect on the activity of the enzyme in homogenates of the muscles, but restored 75% and 91%, respectively, of the activity of cholinesterase in the external surfaces of the muscles exposed to sarin and VX. At the same time, the concentration of acetylcholine required to induce contraction of the muscles was raised to 53.3% and 58.3% of the original threshold concentrations, respectively, for the muscles exposed to sarin and to VX. 

Rajapurkar and Koelle reported that intravenous V at 40 mg/kg, but not at 4 mg/kg, induced reactivation of cholinesterase in the surfaces of cells of the cat s superior cervical ganglion after the animal had been given DFP at 3.7 mg/kg 20 min earlier there was no significant reactivation of the cholinesterase in the ganglion as a whole (after homogenization). These findings suggest that, even when an oxime is able to make contact with the surfaces of nerve cells, it is not able to penetrate into the neurons this is similar to the situation for muscle cells described by Flelsher.94. 

The enzyme choline esterase has been shown to have two binding points on its protein surface for these substances—one site for the quaternary ammonium group and one for X. This enzyme catalyzes the hydrolysis of an ester at the X position. From a consideration of the structure of the (2-chloroethyl)trimethylammonium chloride derivatives which were active as plant growth substances, a similar protein-binding site in the plant has been postulated. This site would have a point of attachment for both the ammonium cation and the X constituent of the molecule. This postulated site in the plant is thus similar, but not identical, to cholinesterase, which is an enzyme not known to occur in plants. There is no direct proof for this hypothetical site in the plant. 

Special interest adheres to the group of cholinesterases (ChE), not only in view of their physiological role in conductive tissues, but also because their specific behavior towards substrates and inhibitors and their high efficiency towards cationic substrates permit exact kinetic measurements. In spite of an enormous amount of experimental work, the exact structure of the active surface of cholinesterases is still controversial [see the review of Whittaker (/)]. The following representation will discuss the results already achieved and point out the many problems in this field still awaiting solution. 

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