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.
Jayanthi
L D
LD
Department of Neurological Sciences, Christian Medical College Hospital, Vellore.
Balasubramanian
A S
AS
D000110:Acetylcholinesterase / Q000302:isolation & purification / Q000378:metabolism*; D000122:Acetylthiocholine / Q000378:metabolism*; D000818:Animals; D001479:Basal Ganglia / Q000201:enzymology*; D007531:Isoflurophate / Q000378:metabolism; D007700:Kinetics; D008251:Macaca; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin
D010446:Peptide Fragments; D007531:Isoflurophate; D000122:Acetylthiocholine; D000110:Acetylcholinesterase; D014357:Trypsin