Species Distribution of Paraoxon‐Resistant Brain Polypeptides Radiolabeled with Diisopropyl Phosphorofluoridate ([3H]DiPF): Electrophoretic Assay for the Aged Polypeptide of [3H]DiPF‐Labelled Neuropathy Target Esterase

Abstract Brain neuropathy target esterase is identified as a paraoxon‐resistant, mipafox‐sensitive esterase that can be labelled with [3H]diisopropyl phosphorofluoridate. During “aging” of the labelled (inhibited) esterase, half the label (one isopropyl group) is transferred to a site (of the same molecular weight in sodium dodecyl sulphate) whence it may be released in volatile form by treatment with alkali. Our previously published procedure for complete extraction in a form suitable for scintillation counting of tritium‐labelled proteins from polyacrylamide gels includes treatment of part‐solubilised gels with alkali. Particles from brain of the hen, pig, sheep, guinea‐pig, and rat were preincubated with paraoxon with or without mipafox, treated with [3H]diisopropyl phosphorofluoridate, and solubilised in sodium dodecyl sulphate. Labelled polypeptides (except from the rat) were separated by electrophoresis. Both mipafox‐sensitive labelling and “volatilisable counts” were located principally in the 155‐kilodalton region, with the residues dispersed throughout the gels. The quantities of paraoxon‐resistant, mipafox‐sensitive labelling sites and of “volatilisable counts” (in pmol/particles from 1 g) were, respectively, 12.2 and 8.65 in hen brain, 9.80 and 6.82 in pig, 8.48 and 5.46 in sheep, 4.46 and 4.01 in guinea‐pig, and 4.91 and 2.08 in rat. The “volatilisable count” assay seems more specific for neuropathy target esterase and is easier and more precise than assays based on differences in labelling of two samples, each subjected to much processing. Hydrolytic activity of particles taken before labelling was measured against phenyl valerate. By comparison with the “volatilisable counts,” the catalytic centre activity of neuropathy target esterase of brain from these species was calculated to be (in the same order) 1.95, 1.95, 2.34, 1.82, and 2.78 ± 105 mol min‐1, respectively.

[1]  C. Meredith,et al.  Neuropathy Target Esterase: Rates of Turnover In Vivo Following Covalent Inhibition with Phenyl Di‐n‐Pentylphosphinate , 1988, Journal of neurochemistry.

[2]  M. Johnson.  Receptor or enzyme: The puzzle of NTE and organophosphate-induced delayed polyneuropathy , 1987 .

[3]  C. Meredith,et al.  Complete extraction in a form suitable for liquid scintillation counting of tritium-labeled proteins from polyacrylamide gels. , 1987, Analytical biochemistry.

[4]  S. Padilla,et al.  An in vitro comparison of rat and chicken brain neurotoxic esterase. , 1986, Fundamental and applied toxicology : official journal of the Society of Toxicology.

[5]  D. G. Williams,et al.  Gel-electrophoretic identification of hen brain neurotoxic esterase, labelled with tritiated di-isopropyl phosphorofluoridate. , 1981, The Biochemical journal.

[6]  B. Clothier,et al.  Rapid aging of neurotoxic esterase after inhibition by di-isopropyl phosphorofluoridate. , 1979, The Biochemical journal.

[7]  M. Johnson,et al.  THE PRIMARY BIOCHEMICAL LESION LEADING TO THE DELAYED NEUROTOXIC EFFECTS OF SOME ORGANOPHOSPHORUS ESTERS , 1974, Journal of neurochemistry.

[8]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[9]  H. S. Anker.  A solubilizable acrylamide gel for electrophoresis , 1970, FEBS letters.

[10]  M. Johnson,et al.  The delayed neurotoxic effect of some organophosphorus compounds. Identification of the phosphorylation site as an esterase. , 1969, The Biochemical journal.

[11]  M. Johnson,et al.  A phosphorylation site in brain and the delayed neurotoxic effect of some organophosphorus compounds. , 1969, The Biochemical journal.