Absence of substrate inhibition and freezing-inactivation of the mosquito acetylcholinesterase are caused by alterations of hydrophobic interactions.
Membrane-bound acetylcholinesterase (AChE) from mosquito showed the characteristic substrate inhibition of this enzyme, but 105,000 x g supernatants of freshly extracted enzyme did not. Addition of chaotropic anions, a freeze-thaw cycle and autolysis of the amphiphilic acetylcholinesterase to its non-amphiphilic derivatives resulted in return of the substrate inhibition feature along with an apparent increment in the enzyme activity. These results suggested that the lipidic environment of the mosquito AChE is temporarily perturbed when extracted. The enzyme is probably trapped in non-sedimenting mixtures composed of endogenous amphiphilic molecules. The occurrence of this phenomenon was not affected by the presence of Triton X-100 and other detergents, either alone or in combination with sodium chloride. Freezing in the presence of strong chaotropic anions (perchlorate, iodide and thiocyanate) caused the irreversible inactivation of the mosquito AChE. Crude and incomplete purified fractions of the enzyme were more sensitive than a more purified preparation. With both the purified AChE and the non-purified AChE, amphiphilic AChE was more freeze labile. Freezing at -10 degrees C enhanced inactivation of non-purified fractions. At this temperature, even weak chaotropic anions (fluoride, chloride and nitrate), while in combination with non-ionic detergents that solubilized mosquito AChE efficiently, reduced the enzyme activity of these fractions. In this case, recovery of the enzyme activity by incubation at 25 degrees C was inversely correlated with the effectiveness of the chaotropic anion. Gel filtration failed to show any change in the hydrodynamic radius of the freezing-inactivated AChE. Therefore, this phenomenon is explained as different degrees of denaturation of the enzyme in direct association with the chaotropic strength. Thus, antichaotropic anions, such as sulfate, should improve the stability of the mosquito acetylcholinesterase during extraction, purification and storage.
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Department of Biochemistry, University of California, Riverside 92521.
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D000110:Acetylcholinesterase / Q000378:metabolism*; D000818:Animals; D000838:Anions; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002712:Chlorides / Q000494:pharmacology; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D009033:Culicidae / Q000201:enzymology*; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005459:Fluorides / Q000494:pharmacology; D005615:Freezing; D006358:Hot Temperature; D007454:Iodides / Q000494:pharmacology; D007700:Kinetics; D009566:Nitrates / Q000494:pharmacology; D010472:Perchlorates / Q000494:pharmacology; D012965:Sodium Chloride; D017670:Sodium Compounds; D012995:Solubility; D013861:Thiocyanates / Q000494:pharmacology
D000838:Anions; D002712:Chlorides; D002800:Cholinesterase Inhibitors; D003902:Detergents; D007454:Iodides; D009566:Nitrates; D010472:Perchlorates; D017670:Sodium Compounds; D013861:Thiocyanates; D012965:Sodium Chloride; C031068:sodium perchlorate; D000110:Acetylcholinesterase; C031760:thiocyanate; D005459:Fluorides