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.