Enzymatic and biochemical characterization of Bungarus sindanus snake venom acetylcholinesterase

This study analyses venom from the elapid krait snake Bungarus sindanus, which contains a high level of acetylcholinesterase (AChE) activity. The enzyme showed optimum activity at alkaline pH (8.5) and 45ºC. Krait venom AChE was inhibited by substrate. Inhibition was significantly reduced by using a high ionic strength buffer; low ionic strength buffer (10 mM PO4 pH 7.5) inhibited the enzyme by 1. 5mM AcSCh, while high ionic strength buffer (62 mM PO4 pH 7.5) inhibited it by 1 mM AcSCh. Venom acetylcholinesterase was also found to be thermally stable at 45ºC; it only lost 5% of its activity after incubation at 45ºC for 40 minutes. The Michaelis-Menten constant (Km) for acetylthiocholine iodide hydrolysis was found to be 0.068 mM. Krait venom acetylcholinesterase was also inhibited by ZnCl2, CdCl2, and HgCl2 in a concentrationdependent manner. Due to the elevated levels of AChE with high catalytic activity and because it is more stable than any other sources, Bungarus sindanus venom is highly valuable for biochemical studies of this enzyme.(AU)

Comparison of two substrates in determination of plasma cholinesterase activity & genetic variability.

Two substrates, acetyl thiocholine iodide ATCI and benzoylcholine chloride BCC were compared for the determination of plasma cholinesterase ChE levels and after incorporation of dibucaine and sodium fluoride in the assay, their usefulness in determining plasma ChE genetic variability was assessed in 64 healthy subjects. With both substrates, plasma ChE levels were found to be in the reference range. However, ATCI could detect only two variants with the usual phenotype UU in 60 of 64 [93.75%] subjects whereas with BCC 6 different groups could be determined. Though both substrates are of equal value in estimation of plasma ChE levels, BCC is definitely superior in determining its genetic variants.

Species variation in the specificity of cholinesterases in human and rat blood samples.

Acetylthiocholine iodide (ATC) as a common substrate in the combined assay of red blood cell cholinesterase (RBC ChE) and butyrylcholinesterase (BuChE) do not provide the accurate individual enzyme activities. Hence, in the present study the two enzyme activities in the same sample were assayed with the help of two different substrate, ATC and butyrylthiocholine iodide (BTC). Specificity of BTC towards BuCHE was found in blood, plasma and serum, while ATC is nonspecifically hydrolysed by both RBC ChE and BuChE. ATC gives significantly higher enzyme activity (P < 0.001) in rat plasma/serum and significantly lower enzyme activity (P < 0.0001; P < 0.001) in human plasma/serum. The possible reasons are discussed for substrate specity in various species in the assay of ChEs.

Improved colorimetric method for cholinesterase activity.

A modified colorimetric method for the estimation of cholinesterase activity has been worked out using two different substrates, acetylthiocholine iodide for total cholinesterase and a specific substrate, butyrylthiocholine iodide for pseudocholinesterase in the same sample. This is a modification of the method described by Voss and Sachsse (1970) wherein acetylthiocholine iodide was used for both total and pseudo cholinesterase activities. The pseudocholinesterase obtained with acetylthiocholine iodide was significantly higher (P < 0.0001) than that with butyrylthiocholine iodide either in whole blood or serum samples. Acetylthiocholine iodide while reacting with pseudocholinesterase in serum or plasma samples might also be interacting with the small quantities of acetylcholinesterase present. It is therefore suggested that butyrylthiocholine iodide and acetylthiocholine iodide may be used to determine pseudocholinesterase and total cholinesterase activities respectively. The use of two substrates with a few more alterations in the experimental conditions increased the validity of this simple and rapid colorimetric method.

Isolation of a tripeptide showing weak acetylthiocholine hydrolysing activity from a soluble form of monkey basal ganglia acetylcholinesterase by limited trypsin digestion.

Acetylcholinesterase was purified from the soluble supernatant of monkey (Macaca radiata) brain basal ganglia by a three-step affinity purification procedure. The purified enzyme showed two major protein bands corresponding to molecular weights of approximately 65 kDa and approximately 58 kDa which could be labelled by [3H]diisopropylfluorophosphate. When the purified enzyme was subjected to limited trypsin digestion followed by gel filtration on Sephadex G-75 or Sephadex G-25 column, a peptide fragment of molecular weight approximately 300 Da having a weak acetylthiocholine hydrolysing activity was isolated. The amino acid sequence analysis of this peptide showed a sequence of Gly-Pro-Ser. When the [3H]DFP labelled enzyme was subjected to limited trypsin digestion and Sephadex G-75 column chromatography, a labelled peptide corresponding to approximately 430 Da was isolated. The kinetics, inhibition characteristics and binding characteristics to lectins of this peptide were compared with the parent enzyme. A synthetic peptide of sequence Gly-Pro-Ser was also found to exhibit acetylthiocholine hydrolysing activity. The kinetics and inhibition characteristics of the synthetic peptide were similar to those of the peptide derived from the purified acetylcholinesterase, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 123700 times less than that of the purified AChE.

Pralidoxime as an insignificant reactivator in severe anticholinesterase (organophosphate insecticide) poisoning.

In acute severe anticholinesterase poisoning by organophosphate compounds, pralidoxime (P-2-AM, pyridine-2-aldoxime methiodide) used in the recommended doses, intravenously, has not been shown to reactivate the inhibited cholinesterase, as evidenced both clinically and biochemically. In vitro studies using pralidoxime iodide up to ten times the recommended concentrations, produced insignificant reactivation of cholinesterases inhibited by the organophosphate insecticide Bidrin (di-methyl-3-hydroxyl-N, N-dimethyl-crotonamide phosphate). This was even so despite prolonged exposure of the inhibited cholinesterases to the oxime. The value of pralidoxime as a reactivator of phosphorylated cholinesterases is therefore in doubt, and should not be used in preference to large doses of atropine and other supportive treatment in poisoning by organophosphate insecticides.