Isolation of a tripeptide (Ala-Gly-Ser) exhibiting weak acetylthiocholine hydrolyzing activity from a high-salt soluble form of monkey diaphragm acetylcholinesterase.
A high-salt soluble form of acetylcholinesterase (AChE) was purified from monkey (Macaca radiata) whole diaphragm by a two step affinity chromatographic procedure using m-aminophenyl trimethylammonium-chloride hydrochloride-Sepharose and procainamide-Sepharose columns. The purified enzyme showed three major protein bands at 80 kDa, 78 kDa and 60 kDa on SDS-gel electrophoresis. [3H]Diisopropyl fluorophosphate ([3H]DFP) labeled enzyme also gave three radioactive peaks corresponding to these three bands. The purified enzyme pretreated with dithiothreitol and subjected to limited trypsin digestion gave a peptide fragment of molecular weight approximately 300 Da showing weak acetylthiocholine hydrolyzing activity as identified by Sephadex G-25 gel filtration. Sequence analysis showed that the active peptide fragment was a tripeptide with the sequence Ala-Gly-Ser. When the purified AChE was labeled with [3H]DFP, digested with trypsin and subjected to Sephadex G-25 chromatography, a radioactive peak that would correspond to the tripeptide fragment was seen. The kinetics, inhibition characteristics and binding characteristics to lectins of the active peptide fragment was compared with the parent enzyme. A synthetic peptide of sequence Ala-Gly-Ser was also found to exhibit acetylthiocholine hydrolyzing activity. The kinetics and inhibition characteristics of the synthetic peptide was similar to those of the peptide derived from the purified enzyme, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 29480 times less than that of the purified AChE.
Jayanthi
L D
LD
Department of Neurological Sciences, Christian Medical College Hospital, Vellore, India.
Balasubramanian
A S
AS
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000302:isolation & purification; D000122:Acetylthiocholine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003964:Diaphragm / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007531:Isoflurophate; D008255:Macaca radiata; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000302:isolation & purification*; D012492:Salts; D012995:Solubility; D014357:Trypsin
D009842:Oligopeptides; D012492:Salts; D007531:Isoflurophate; D000122:Acetylthiocholine; C076328:alanyl-glycyl-serine; D000110:Acetylcholinesterase; D014357:Trypsin