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Meredith C, Johnson MK: Species distribution of paraoxon-resistant brain polypeptides radiolabelled with diisopropyl phosphorofluoridate ([3H] DiPF): electrophoretic assay for the aged polypeptide of [3H] DiPF-labelled neuropathy target esterase. J Neurochem. 1989 Apr;52(4):1248-52.
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.(ABSTRACT TRUNCATED AT 250 WORDS) |
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