Neurofilament phosphorylation and [125I]Calmodulin binding by Ca2+/calmodulin-dependent protein kinase in the brain subcellular fractions of diisopropyl phosphorofluoridate (DFP)-treated hen |
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Authors: | Ram P Gupta Mohamed B Abou-Donia |
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Institution: | (1) Department of Pharmacology, Duke University Medical Center, P.O. Box 3813, 27710-0001 Durham, North Carolina |
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Abstract: | Diisopropyl phosphorofluoridate (DFP) produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in humans and sensitive animal species, e.g., adult chicken. The chickens were sacrificed 18 days after a single dose of DFP (1.7 mg/kg, sc.), which produced severe ataxia or paralysis in 10–14 days. We studied Ca2+/calmodulin-dependent in vitro neurofilament phosphorylation by the brain subcellular fractions of control and DFP-treated hens. There was enhanced phosphorylation of all three NF subunits by the brain supernatant of treated hens. This was accompanied by enhanced autophosphorylation of both Ca2+/CaM-dependent protein kinase II (CaM-kinase II) subunits and increased calmodulin binding using either125I-CaM or biotinylated calmodulin to only subunit without concomitant increase in the amount of this enzyme. This enhanced phosphorylation of neurofilament subunits was completely and partially inhibited by mastoparan and KN-62, respectively. There was no alteration in the distribution of CaM-kinase II activity in treated hens and the activity was not related to its concentration in different subcellular fractions. The difference in125I-CaM binding to CaM-kinase II subunit in the brain supernatants of control and DFP-treated hens was not altered by its phosphorylation or dephosphorylation. The increased CaM-kinase II activity in the soluble fraction of DFP-treated hen brain may be involved in the aberrant phosphorylation of axonal neurofilaments, and thus play a role in OPIDN.Abbreviations CaM
calmodulin
- CaM-kinase II
Ca2+/calmodulin-dependent protein kinase II
- DFP
diisopropyl phosphorofluoridate
- ECL
enhanced chemiluminescence
- EDTA
ethylenediaminetetraacetic acid
- EGTA
ethylene glycol-bis( -aminoethyl ether)N,N,N ,N -tetraacetic acid
- MAP-2
microtubule-associated protein-2
- MBP
myelin basic protein
- OPIDN
organophosphorus ester-induced delayed neurotoxicity
- PIPES
1,4-piperazinediethanesulfonic acid
- PMSF
phenylmethylsulfonyl fluoride
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- TCA
trichloroacetic acid |
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Keywords: | Neurofilament phosphorylation Ca2+/calmodulin-dependent protein kinase calmodulin-binding hen diisopropyl phosphorufluoridate brain |
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