Kinetic evidence of multiple reversible cholinesterases based on inhibition by organophosphates.

Inhibition of serum choline&erase (ChE) and erythrocyte acetylcholinesterase by diisopropylphosphorofluoridate and amiton did not follow first order kinetics. Curving of the rate plots was concave upward and varied greatly. An equation was derived on the assumption that curving reflected inhibition of a multiple enzyme system. The curves obtained with partially purified horse ChE were then resolved into four linear components at 5” and into three linear components with an indication of a fourth at 25’. Similar results were obtained with human IV-6-3 ChE and bovine erythrocyte acetylcholinesterase. Each resolved component represented the first order rate plot for the inhibition of one form of ChE, indicating that four forms were present in each preparation. The phosphorylation (kz) and binding constants (KJ of the various forms of horse and human ChE inhibited by amiton at 5” and of horse ChE inhibited by diisopropylphosphorofluoridate at 25” were obtained and were found to vary significantly. For example, the kt values of horse ChE inhibited by amiton at 5” varied from 48.8 f 2.2 min-l to 0.0028 =t 0.0003 min+while Gvariedfrom 7.6 f 1.2 x lop6 M to 3.2 f 1.5 X 1O-7 M. Binding increased as kz decreased with one exception. Since cholinesterases are probably agglomerates of subunits, this indicated that the kinetic properties of the various forms may differ profoundly with the state of agglomeration. However, additional results also indicated that not every inhibitor nor inhibition at every concentration is capable of bringing out these differences. The intercepts at f = 0 of the linear first order plots were independent of the inhibitor used and of its concentration and provided a criterion by which shifts in the relative concentrations of the various forms could be followed. Changes in either temperature or the ChE concentration changed the relative concentrations of the multiple forms,