Reversibility of the inhibition of true cholinesterase by physostigmine

Abstract

1. 1. The kinetics of a true cholinesterase preparation from rat brain were studied. Acetyl-β-methylcholine (amechol) was used as a substrate. It was found that, at a constant concentration of enzyme, the reaction is of a unimolecular type when the concentration of substrate is lower than 0.01 M; when the substrate concentration rises above 0.01 M the reaction assumes a zero order character. 2. 2. When the reaction velocity is determined in presence of physostigmine, it is found that a progressive inhibition takes place during the test, which is slowed down, but never reversed by the substrate amechol. 3. 3. When the enzyme is incubated for varying periods, prior to the activity test, with the same low concentration of physostigmine, it is found that the degree of inhibition increases with the duration of the incubation period. It is found that also under these conditions, in presence of a low concentration of physostigmine, the inhibition is progressive and is not reversed by the substrate, when this is added after the incubation period. In these experiments amechol was used as a substrate. 4. 4. Dialysis reverses the inhibition by physostigmine of true cholinesterase towards acetylcholine in 4 hours. The reversal of the inhibition of pseudo cholinesterase appears to proceed more slowly. 5. 5. Washing out of rat brain suspensions previously inhibited with physostigmine does not cause reversal of the inhibition. 6. 6. It is not possible to reverse part of the inhibition acutely by means of dilution after ten minutes of incubation. 7. 7. It is concluded that no competitive reversal by substrate of physostigmine inhibition takes place, but that it is possible to reverse this inhibition by dialysis for several hours. Within shorter observation periods no reversal is demonstrable by dialysis or dilution using acetylcholine as a substrate except after very short periods of incubation. 8. 8. It is suggested that, as a result of incubation with physostigmine, two types of inhibition of cholinesterase may occur simultaneously; one towards amechol, the other towards acetylcholine. The physostigmine inhibition probably affects two different active groups, both present in our preparation of true cholinesterase.