Rapid aging of neurotoxic esterase after inhibition by di-isopropyl phosphorofluoridate

Abstract

1. It was proposed [Johnson (1974) J. Neurochem. 23, 785–789] that an essential step in the genesis of delayed neuropathy caused by some organophosphorus esters was aging of phosphorylated neurotoxic esterase, involving generation of a charged monosubstituted phosphoric acid residue on the protein. 2. Neurotoxic esterase of hen brain was inhibited with di-isopropyl phosphorofluoridate either unlabelled or mixed-labelled with ³H and ³²P. 3. Reactivation of inhibited enzyme by KF was possible only immediately after a brief inhibition:aging at pH8.0 and 37°C occurred with a half-life of about 2–4min. 4. When the radiolabelled enzyme was studied no loss of label was observed during the expected aging period, but a change in the nature of the bound radioisotopes occurred (half-life=3.25min). 5. Alkaline hydrolysis of labelled enzyme liberated di-isopropyl phosphate at early times after labelling, but increasing amounts of monoisopropyl phosphate plus a volatile tritiated compound (possibly propan-2-ol) at later times. 6. Treatment of labelled enzyme with KF released di-isopropyl phosphate and caused reactivation of enzyme to similar degrees. It is concluded that the chemical change from di-isopropyl phosphoryl-enzyme to mono-isopropyl phosphoryl-enzyme and the loss of reactivatibility are related. 7. The rate of aging is similar at pH5.2, 6.5 and 8. Aging is unaffected by addition of reduced glutathione and imidazole at pH5.2 or 8, and none of the transferred ³H is trapped by these reagents. The mechanism of aging must be different from the better-known dealkylation aging of the cholinesterases.