Gel-electrophoretic identification of hen brain neurotoxic esterase, labelled with tritiated di-isopropyl phosphorofluoridate

Publisher Website
Google Scholar
Abstract
The particulate fraction from hen brain was labeled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated by polyacrylamide-gel electrophoresis. Four radioactive protein bands (1-4) of MW 155,000, 92,000, 60,000 and 30,000 were resolved. Most of the labeling of bands 2, 3 and 4 was inhibited by preincubation with Paraoxon. The residue in band 4 was sensitive to pH 5.2. Successive treatments with Paraoxon and pH 5.2 resulted in the abolition of bands 3 and 4. Bands 1 and 2 contained 1 and 2 polypeptides, respectively, whose labeling was sensitive to Mipafox, but 1, in band 2, was sensitive to higher concentrations of Paraoxon. The concentrations of the other 2 polypeptides were 6.7 and 1.95 pmol of DiPF bound/g of brain in bands 1 and 2, respectively. Both were as sensitive to Mipafox as neurotoxic esterase and were also sensitive to phenyl benzylcarbamate. 4-Nitrophenyl di-n-pentylphosphinate given in vivo inhibited neurotoxic esterase and the labeling of the band-1 polypeptide by 82 and 84%, respectively, but inhibited the labeling of the band 2 polypeptide by 51%. The phosphinate in vitro produced 98% inhibition of the labeling of the band-1 polypeptide, with 26% inhibition of the band-2 polypeptide, under conditions sufficient to inhibit neurotoxic esterase totally. Both neurotoxic esterase and the band-1 polypeptide were found in the forebrain at 1.74-fold their concentration in the rest of the brain, whereas the band-2 polypeptide was uniformly distributed. The Mipafox-sensitive polypeptide in band 1 is the [3H]-DiPF-labeled active-site subunit of neurotoxic esterase. The catalytic-center activity of the enzyme for phenyl valerate hydrolysis was 2.6 .times. 105 min-1.