DNA sequence of butyrylcholinesterase from the rat: expression of the protein and characterization of the properties of rat butyrylcholinesterase.
The rat is the model animal for toxicity studies. Butyrylcholinesterase (BChE), being sensitive to inhibition by some organophosphorus and carbamate pesticides, is a biomarker of toxic exposure. The goal of this work was to characterize the purified rat BChE enzyme. The cDNA sequence showed eight amino acid differences between the active site gorge of rat and human BChE, six clustered around the acyl binding pocket and two below the active site serine. A prominent difference in rat was the substitution of arginine for leucine at position 286 in the acyl pocket. Wild-type rat BChE, the mutant R286L, wild-type human BChE, and the mutant L286R were expressed in CHO cells and purified. Arg286 was found responsible for the resistance of rat BChE to inhibition by Triton X-100. Replacement of Arg286 with leucine caused the affinity for Triton X-100 to increase 20-fold, making it as sensitive as human BChE to inhibition by Triton X-100. Wild-type rat BChE had an 8- to 9-fold higher K(m) for the positively charged substrates butyrylthiocholine, acetylthiocholine, propionylthiocholine, benzoylcholine, and cocaine compared with wild-type human BChE. Wild-type rat BChE catalyzed turnover 2- to 7-fold more rapidly than human BChE, showing the highest turnover with propionylthiocholine (201,000 min(-1)). Human BChE does not reactivate spontaneously after inhibition by echothiophate, but rat BChE reactivates with a half-life of 4.3hr. Human serum contains 5mg/L of BChE and 0.01mg/L of AChE. Male rat serum contains 0.2mg/L of BChE and approximately 0.2mg/L of AChE.
Eppley Institute, 986805, University of Nebraska Medical Center, Omaha, NE 68198-6805, USA.
Andreea Ticu
AT
Boeck
Lawrence M
LM
Schopfer
Oksana
O
Lockridge
D000110:Acetylcholinesterase / Q000097:blood; D000122:Acetylthiocholine / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001588:Benzoylcholine / Q000378:metabolism; D002091:Butyrylcholinesterase / Q000097:blood / Q000235:genetics* / Q000378:metabolism; D002092:Butyrylthiocholine / Q000378:metabolism; D003042:Cocaine / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis; D004456:Echothiophate Iodide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017830:Octoxynol / Q000494:pharmacology*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013860:Thiocholine / Q000031:analogs & derivatives* / Q000378:metabolism; D014020:Tissue Extracts
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D014020:Tissue Extracts; D001588:Benzoylcholine; C009063:propionylthiocholine; D000122:Acetylthiocholine; D002092:Butyrylthiocholine; D013860:Thiocholine; D017830:Octoxynol; D004456:Echothiophate Iodide; D000110:Acetylcholinesterase; D002091:Butyrylcholinesterase; D003042:Cocaine