Organophosphates, such as tetraethyl pyrophosphate (TEPP) and diisopropyl phosphorofluoridate (DFP), which have a dialkylphosphato group, form with human plasma cholinesterase (also called pseudo-or butyro-cholinesterase) two types of phosphorylated enzyme which possess different stabilities of the enzyme-phosphorus linkage (Hobbiger, 1955). Only phosphorylated enzyme J, which is formed initially, can be readily reactivated by nucleophilic reagents such as nicotinhydroxamic acid methiodide (NHAM). Phosphorylated enzyme I then changes into phosphorylated enzyme II, most probably by transphosphorylation. With DFP or diisopropyl p-nitrophenylphosphate (D 600) the rate of transphosphorylation is faster than with TEPP or diethyl p-nitrophenylphosphate (E 600) and at 370 C. and a pH of 7.45 approximately 90% of the inhibited enzyme is converted into phosphorylated enzyme II within 1 hour.Since the hydrolysis of acetylcholine in vivo is mainly accomplished by true cholinesterase (also called aceto-cholinesterase) the formation of a phosphorylated true cholinesterase which cannot be reactivated by nucleophilic reagents could seriously affect the therapeutic usefulness of NHAM and other more potent substances. The experiments on human plasma cholinesterase were therefore extended to human and bovine true cholinesterases which had been inhibited by an organophosphate containing either a diethyl-or diisopropylphosphato group and the findings are reported in this paper. In addition to NHAM, ammonium molybdate and pyridine-2-aldoxime methiodide, a potent reactivator of phosphorylated electric eel cholinesterase and phosphorylated human and rat true ch6lin-esterases (Wilson and Ginsburg, 1955;and Childs, Davies, Green, and Rutland, 1955), were used for enzyme reactivation. The results obtained with all these reactivators show that the organophosphates form two types of phosphorylated enzyme with human and bovine true cholinesterases which thus behave like plasma cholinesterase. Only the initial form (phosphorylated enzyme l) can readily be reactivated by the nucleophilic reagents. Transphosphorylation-formation of the truly irreversible phosphorylated enzyme Il-occurs both in vitro and in vivo, and the rate of transphosphorylation is dependent upon pH.
METHODSAU experiments were carried out in the Warburg apparatus.Cholinesterase activity was determined by the manometric technique using 0.01 M-acetylcholine chloride as substrate for human and bovine true cholinesterases and 0.02 M-(±)-acetyl-f-methylcholine chloride as substrate for the true cholinesterases of whole rabbit blood. The procedures adopted for determination of enzyme activity and reactivation were the same as those described in an earlier publication (Hobbiger, 1955).Except when otherwise stated, the gas phase consisted of 95% N2+5% CO2 and a solution containing 0.025 M-NaHCO3, 0.075 M-NaCl, 0.075 M-KCl, 0.04 M-MgCl2 and 0.1% bovine plasma albumin (Armour Laboratories) was used for all dilutions and as the medium during inhibition, reactivation and determination ...