Purification and characterization of an acetylcholinesterase from the infective juveniles of Heterorhabditis bacteriophora.
Acetylcholinesterases (AChEs) have been estimated in the infective juveniles (IJs) of eight different strains of heterorhabditid nematodes. The enzyme content ranged from 45.6 to 421.3 units/10(5) IJs with specific activity 34.0 to 82.6 units/mg protein. The isoenzyme patterns revealed the existence of two-slow-moving isoforms. Heterorhabditis bacteriophora AChE1A has been purified from the IJs of the heterorhabditid nematode strain of the highest enzymatic activity to homogeneity by ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and DEAE-Sepharose. The specific activity of the purified enzyme was 1378.1 units/mg protein with purification fold 17.5 over crude extract. The enzyme has a pH optimum at 7.5. The optimum temperature for enzyme activity and stability was 35 degrees C. The activation energy was calculated to be 9.0 kcal/mol. The enzyme hydrolyzes acetylthiocholine (AcSCh), propionylthiocholine (PrSCh), S-butyrylthiocholine (BuSCh) and benzoylthiocholine (BzSCh) iodides with relative rate 100, 74.6, 41.7 and 22.2%, respectively. It displayed an apparent Michaelis-Menten behavior in the concentration range from 0.1 to 2 mM for the three former substrates with Km values 0.27, 0.42 and 0.59 mM, respectively. H. bacteriophora ChE1A is an AChE since it hydrolyzed AcSChI at higher rate than the other substrates and displayed excess substrate inhibition with AcSChI at concentrations over 2 mM. It was inhibited by eserine and BW284C51, but not by iso-OMPA. Its biochemical properties were compared with those reported for different species of insects as target hosts for heterorhabditid nematodes and animal parasitic nematodes.
Mohamed
Magda A
MA
Molecular Biology Department, National Research Centre, Cairo, Egypt. magdasoroor@yahoo.com
Abdel-Gawad
Amira S
AS
Ghazy
Abd-Elhady M
AE
D000110:Acetylcholinesterase / Q000302:isolation & purification* / Q000378:metabolism*; D000122:Acetylthiocholine / Q000378:metabolism; D000818:Animals; D001588:Benzoylcholine / Q000031:analogs & derivatives / Q000378:metabolism; D002092:Butyrylthiocholine / Q000378:metabolism; D006868:Hydrolysis; D008018:Life Cycle Stages / Q000502:physiology*; D017172:Rhabditoidea / Q000201:enzymology*; D013379:Substrate Specificity; D013860:Thiocholine / Q000031:analogs & derivatives / Q000378:metabolism
C518182:benzoylthiocholine; D001588:Benzoylcholine; C009063:propionylthiocholine; D000122:Acetylthiocholine; D002092:Butyrylthiocholine; D013860:Thiocholine; D000110:Acetylcholinesterase