Interactions of rat brain acetylcholinesterase with the detergent Triton X-100 and the organophosphate paraoxon.
Inhibition of the critical enzyme acetylcholinesterase (E.C. 3.1.1.7) with subsequent cholinergic crisis is the mechanism of acute toxicity of the organophosphorus insecticides (B. E. Mileson et al., 1998, Toxicol. Sci.41, 8-20). Consequently, measurement of acetylcholinesterase activity is important for evaluating the mammalian toxicity of this commonly used class of insecticides. While mammalian acetylcholinesterase activity has often been determined in tissue homogenates in the presence of the nondenaturing detergent Triton X-100 at a concentration of 1%, the potential actions of this detergent on the activity of this critical enzyme are not understood. In the current study, homogenization of rat brain in buffer containing 1% Triton X-100 slightly elevated the (app)V(max) for hydrolysis of acetylthiocholine, without affecting the (app)K(m) or the (app)K(ss). However, the presence of both 1% Triton X-100 and paraoxon (at concentrations of 5 nM-100 nM) resulted in complex kinetic interactions with acetylcholinesterase, as evidenced by a curvilinear secondary plot for determination of the (app)k(i). These results suggest that measurement of acetylcholinesterase activity in the presence of up to 1% Triton X-100, but in the absence of oxon, should pose no problems with regard to data interpretation, provided it is recognized that the detergent slightly elevates activity. However, measurement of acetylcholinesterase activity after enzyme was exposed simultaneously to Triton X-100 and oxon could be problematic. Caution is warranted when interpreting data where acetylcholinesterase activity was determined under such conditions since in the presence of 1% Triton X-100, the capacity of oxon to inhibit acetylcholinesterase might change as a function of oxon levels.
Rosenfeld
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C
Department of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, New Jersey 07103, USA.
Kousba
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A
Sultatos
L G
LG
D000109:Acetylcholine / Q000494:pharmacology*; D000110:Acetylcholinesterase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D003902:Detergents / Q000378:metabolism*; D004347:Drug Interactions; D007306:Insecticides / Q000378:metabolism*; D008297:Male; D008954:Models, Biological; D017830:Octoxynol / Q000378:metabolism*; D010261:Paraoxon / Q000378:metabolism*; D010599:Pharmacokinetics; D051381:Rats; D017207:Rats, Sprague-Dawley
D003902:Detergents; D007306:Insecticides; D017830:Octoxynol; D000110:Acetylcholinesterase; D000109:Acetylcholine; D010261:Paraoxon