ADP-ribosylation of microtubule proteins as catalyzed by cholera toxin.
Incubation of purified rat brain tubulin with cholera toxin and radiolabeled [32P] or [8-3H]NAD results in the labeling of both alpha and beta subunits as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of these protein bands with snake venom phosphodiesterase resulted in quantitative release of labeled 5'-AMP, respectively labeled with the corresponding isotope. Two-dimensional separation by isoelectric focusing and SDS-PAGE of labeled and native tubulin revealed that labeling occurs at least in four different isotubulins. The isoelectric point of the labeled isotubulins was slightly lower than that of native purified tubulin. This shift in mobility is probably due to additional negative charges involved with the incorporation of ADP-ribosyl residues into the tubulin subunits. SDS-PAGE of peptides derived from [32P]ADP-ribosylated alpha and beta tubulin subunits by Staphylococcus aureus protease cleavage showed a peptide pattern identical with that of native tubulin. Microtubule-associated proteins (MAP1 and MAP2) of high molecular weight were also shown to undergo ADP-ribosylation. Incubation of permeated rat neuroblastoma cells in the presence of [32P]NAD and cholera toxin results in the labeling of only a few cell proteins of which tubulin is one of the major substrates.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001923:Brain Chemistry; D002772:Cholera Toxin / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008869:Microtubule-Associated Proteins; D009243:NAD / Q000378:metabolism; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D012697:Serine Endopeptidases; D014404:Tubulin / Q000378:metabolism*
D008869:Microtubule-Associated Proteins; D009702:Nucleoside Diphosphate Sugars; D011506:Proteins; D014404:Tubulin; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase