Abstract
Peptidylarginine deiminase (proteinarginine iminohydrolase, EC 3.5.3.15) converted some arginine residues to citrulline residues in soluble vimentin, in a micromolar Ca2+-dependent manner and resulted in the loss of polymerization competence of the intermediate filament protein. When about 8 mol of residues/mol of vimentin were deiminated, there was a complete loss of filament forming ability. This enzyme also deiminated vimentin filaments which had been polymerized, and deimination of vimentin filaments resulted in filament disassembly. Similar results were obtained with other intermediate filaments such as desmin and glial filaments. High performance liquid chromatography and amino acid analyses of lysine-specific protease-generated fragments from deiminated vimentin (about 8 mol of citrulline/mol of vimentin) showed a differential deimination of three structural domains. The head domain was predominant. These observations suggest that the head domain strongly influences integrity of the intermediate filament.
Highlights
Peptidylarginine deiminase converted some arginine residues to citrulline residues in soluble vimentin, ina micromolar Ca2+-dependentmanner and resulted in the loss of polymerization competence of the intermediate filament protein
To study regulatory mechanisms governing assembly-disassembly of intermediate filaments, we investigated specific phosphorylation events which modulate the assembly of vimentin and desmin mlolecules into intermediate filaments[10, 11].Vimentin and desmin are excellent in uitro substrates for
2) These reactions were regulated by the presence of micromolar Ca2+concentration in the reaction mixture.3) The amino-terminalnon-a-helical head domain of vimentin was the predominant target of peptidylarginine deiminase
Summary
IntermediateFilament Proteins-Vimentin from Furth’s murine mastocytoma cells, desminfromchicken gizzard, andGFAP from bovine brain were purified in 7 M urea, using standard procedures [10,11, 24,25,26,27]. Deimination of Intermediate Filament Proteins-Unless otherwise Fragmentation of Native and Deiminuted Vimentin-Native and specified, intermediate filament protein(s0.5 mg/ml) weredeiminated deiminatedvimentin werereduced andS-pyridylethylated by the by incubation, ina final volume of 50 pl, with 1.25 pg/ml (0.78 units/ rnl) peptidylarginine deiminase, 25 mM Tris-HCI buffer, pH 7.0, 1 method of Hermondson et al [31].Five mg of each pyridylethylated vimentin was digested with 8 pg of lysyl endopeptidase (Wako Pure taamitddMedIysiilondtailciroatgunhtiibionooanifttnhi8oeorfnepdD1ietpeooieimflmr,i1ioinan7dana8sdtameedtwMC2aaV5sE*i+“oGmCb-TeEt,naAGttihin.Tnee-AdVre,ifamracosetminoitninandswiccaaadslteeetidedm-r.uminApianfrttaeeetdarecdttwihoiebtnhyamptphpiexerp-o-priecptrC-heqeHhvitusieeic9mlrdi.sa0ibiegcc,r-eiaacpdsltoh.teiLnadoEttsndalweu.icw,nitotiiheoOlnurngesm1aw2s4kne%a(apMsS,a(ecJruvnaaar/srptverheaa)iduenaPad)bcatyeiokn3tuoH0tn7Pw“i0tLCr0itiCOhlfpeo1D(craHSoo1f-ni661tt50a1ah0-c5i.mnh1mPii-ineNMnmgp,oltiTidn40dere..e6l1iass%6r-Xo3Hgb8trCrt)a1iaIdf5oilin0nubeeounamdrftfoCmebaoryM-)f, ture. The centrifugation procedurecould be used to isolate the deiminatedvimentin.Sodiumchloridewasadded to a scaled-up reaction mixture, to give 150 mM, and the preparationwas incubated for a further 1 h a t 37 “C. After removalof filament virnentinby high speed centrifugation (10,000 X g, 30rnin), thesupernatantwas
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