The modification of cholinesterase activity by 5,5'-dithiobis-(2-nitrobenzoic acid) included in the coupled spectrophotometric assay. Evidence for a non-catalytic substrate-binding site.
1. Compared with the acetylcholinesterase assay carried out in the absence of a dithiol, the presence of 5,5'-dithiobis-(2-nitrobenzoic acid) caused marked activation, 6,6'-dithiodinicotinic acid and 2,2'-dithiobis-(5-nitropyridine) less so and 2,2'-dithiodipyridine (aldrithiol-2) had no effect at all. Measurements are further complicated in that the 5-thio-2-nitrobenzoate ion also appears to interact with the enzyme, resulting in slightly lowered absorbance values. 2. Acetylthiocholine competes for the 5,5'-dithiobis-(2-nitrobenzoic acid)-binding site so that activation is essentially eliminated by saturating concentrations of substrate. The presence of the dithiol decreases the K(m) value of acetylthiocholine. 3. Similar results were obtained with pseudocholinesterase. However, with butyrylthiocholine clear activation was still observed under V(max.) conditions in addition to K(m) being lowered. 4. All the data yielded Hill coefficients of 1 and analysis of the results leads to the conclusion that activation results from the dithiol being bound to a site on the subunit that is actively catalysing ester hydrolysis. 5. The use of aldrithiol-2 is recommended for kinetic work where absolute quantitative measurements are required.
D001565:Benzoates / Q000494:pharmacology*; D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002800:Cholinesterase Inhibitors; D002802:Cholinesterases / Q000097:blood* / Q000378:metabolism; D004220:Disulfides / Q000494:pharmacology; D004789:Enzyme Activation; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D009539:Nicotinic Acids / Q000494:pharmacology; D009574:Nitro Compounds / Q000494:pharmacology; D011485:Protein Binding; D011725:Pyridines / Q000494:pharmacology; D012995:Solubility; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013501:Surface-Active Agents
D001565:Benzoates; D002800:Cholinesterase Inhibitors; D004220:Disulfides; D009539:Nicotinic Acids; D009574:Nitro Compounds; D011725:Pyridines; D013501:Surface-Active Agents; D002802:Cholinesterases