Chemical Reactivation of Phosphorylated Human and Bovine True Cholinesterases

Abstract

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 ...

Acetylcholinesterase in the fast extraocular muscle of the mouse by light and electron microscope autoradiography.

“…The conditions were also designed to eliminate any significant effect of "ageing" on the quantitative results by keeping the time lapse between the DFP and the 2-PAM incubation to <1 h. ("Ageing" is a process whereby dealkylation of the organophosphorous inhibited enzymes occurs with time and prevents reactivation by 2-PAM. The accumulated evidence from several studies [5,9,11,14,21,22,23] leads to the conclusion that the half-life for ageing of AChe inactivated with DFP is longer than for butyrylcholinesterase (BuChe), is on the order of 10-35 h at room temperature, and is temperature-dependent. )…”

Effects of quaternary ammonium compounds on choline transport in red cells

“…Since inhilbition of acetylcholinesterase by organo-phosphorus compounds is progressive, another attempt at blocking the cholinesterase activity was made by incubating erythrocytes overnight with 10-5M DFP. This treatment reduces the cholinesterase activity to about 3-4% of the original value (Burgen & Hobbiger, 1951) and leads to the conversion of reactivatable into non-reactivatable phosphorylated acetyloholinesterase (Hobbiger, 1956). However, it turned out that this treatment also reduced The affinity constants are the reciprocals of the dissociation constants.…”

Quantitative Studies on Enzymes in Structures in Striated Muscles by Labeled Inhibitor Methods

“…Since this process would reduce the radioactivity liberated by 2-PAM in our experiments, an attempt was made to measure the rate of ageing in our conditions of work . From three mice, the strip of endplates from the entire diaphragm was Hobbiger (1956) and of Davies and Green (1956) .…”