Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.1.8 (cholinesterase)
 12,691 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three-dimensional structure, a fragment of human butyrylcholinesterase (BuChE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed. Immunoblot analysis revealed that the clone-produced BuChE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE). Rabbit polyclonal antibodies prepared against the purified clone-produced BuChE polypeptides interacted in immunoblots with denatured serum BuChE as well as with purified and denatured erythrocyte AChE. In contrast, native BuChE tetramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody-enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation. Furthermore, both AChE and BuChE dimers from muscle extracts, but not BuChE tetramers from muscle, interacted with these antibodies. To reveal further whether the anti-cloned BuChE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve. Muscle fibers incubated with the antibodies and with 125I-Protein A were subjected to emulsion autoradiography, followed by cytochemical ChE staining. The anti-cloned BuChE antibodies, as well as anti-Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not. These findings demonstrate that the various forms of human AChE and BuChE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.
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PMID:Cross-homologies and structural differences between human cholinesterases revealed by antibodies against cDNA-produced human butyrylcholinesterase peptides. 246 May 89

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.
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PMID:Cholinesterase-like catalytic antibodies: reaction with substrates and inhibitors. 1127 56