Cholinesterase-like catalytic antibodies: reaction with substrates and inhibitors.
We have previously described a catalytic monoclonal antibody, raised against acetylcholinesterase (AChE) and capable of hydrolysing acetylthiocholine. Here, we describe two more such antibodies. All three antibodies were raised against the same antigen, human erythrocyte AChE, a commercial product purified using the cholinesterase anionic site inhibitor, tetramethylammonium. IgG was purified on Protein A-Sepharose, and lack of contamination with AChE or butyrylcholinesterase (BChE) was demonstrated on sucrose density gradients and immunoassay of the fractions. The antibodies recognised AchE and were capable of hydrolysing acetylthiocholine and the larger butyrylthiocholine substrate, and were inactivated by phenylmethylsulphonyl fluoride (PMSF), indicating a serine residue in the active site. K(m), K(cat), K(cat)/K(uncat) and K(cat)/K(m) values were obtained for both substrates. The active sites of the antibodies were probed with anti-cholinesterases known to react with the active and anionic sites of acetyl- and BChE, and the peripheral anionic site of AChE. The antibodies were inactivated to varying degrees by the BChE inhibitors iso-OMPA, ethopropazine and tetracaine, indicating a less sterically constrained site than AChE and the lack of an acyl-binding pocket. They were also partially inhibited by the AChE-specific inhibitors, BW284c51 and propidium. No peripheral anionic site, as seen in AChE, was observed, shown by the almost complete lack of reaction with fasciculin. All three antibodies appear to have structures resembling the anionic sites of the cholinesterases, seen by their inhibition by quaternary and tricyclic compounds. Further work is required to determine whether the catalytic activity shown by these antibodies is germline-encoded, or is the result of complexation of the antigen with an inhibitor at a peripheral site.
Johnson
G
G
Department of Pediatric Surgery, Medical Faculty, University of Stellenbosch, PO Box 19063, 7505, Tygerberg, South Africa. gjo@maties.sun.ac.za
Moore
S W
SW
D000122:Acetylthiocholine / Q000378:metabolism; D017151:Antibodies, Catalytic / Q000187:drug effects / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000187:drug effects / Q000378:metabolism; D000918:Antibody Specificity; D002092:Butyrylthiocholine / Q000378:metabolism; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D002802:Cholinesterases / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D010640:Phenothiazines / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D013379:Substrate Specificity; D013748:Tetracaine / Q000494:pharmacology; D013770:Tetraisopropylpyrophosphamide / Q000494:pharmacology
D017151:Antibodies, Catalytic; D000911:Antibodies, Monoclonal; D002800:Cholinesterase Inhibitors; D010640:Phenothiazines; D013748:Tetracaine; D000122:Acetylthiocholine; D002092:Butyrylthiocholine; D013770:Tetraisopropylpyrophosphamide; D010664:Phenylmethylsulfonyl Fluoride; C084820:profenamine; D002802:Cholinesterases