Characterization of chemical inhibitors of brefeldin A-activated mono- ADP-ribosylation

Roberto Weigert, Antonino Colanzi, Alexander Mironov, Roberto Buccione, Claudia Cericola, Maria Gina Sciulli, Giovanna Santini, Silvio Flati, Aurora Fusella, Julie G. Donaldson, Maria Di Girolamo, Daniela Corda, Maria Antonietta De Matteis, Alberto Luini

Research output: Contribution to journalArticlepeer-review


Brefeldin A, a toxin inhibitor of vesicular traffic, induces the selective mono-ADP-ribosylation of two cytosolic proteins, glyceraldehyde-3- phosphate dehydrogenase and the novel GTP-binding protein BARS-50. Here, we have used a new quantitative assay for the characterization of this reaction and the development of specific pharmacological inhibitors. Mono-ADP- ribosylation is activated by brefeldin A with an EC50 of 17.0 ± 3.1 μg/ml, but not by biologically inactive analogs including a brefeldin A stereoisomer. Brefeldin A acts by increasing the V(max) of the reaction, whereas it does not influence the K(m) of the enzyme for NAD+ (154 ± 13 μM). The enzyme is an integral membrane protein present in most tissues and is modulated by Zn2+, Cu2+, ATP (but not by other nucleotides), pH, temperature, and ionic strength. To identify inhibitors of the reaction, a large number of drugs previously tested as blockers of bacterial ADP- ribosyltransferases were screened. Two classes of molecules, one belonging to the coumarin group (dicumarol, coumermycin A1, and novobiocin) and the other to the quinone group (ilimaquinone, benzoquinone, and naphthoquinone), rather potently and specifically inhibited brefeldin A-dependent mono-ADP- ribosylation. When tested in living cells, these molecules antagonized the tubular reticular redistribution of the Golgi complex caused by brefeldin A at concentrations similar to those active in the mono-ADP-ribosylation assay in vitro, suggesting a role for mono-ADP-ribosylation in the cellular actions of brefeldin A.

Original languageEnglish
Pages (from-to)14200-14207
Number of pages8
JournalJournal of Biological Chemistry
Issue number22
Publication statusPublished - May 30 1997

ASJC Scopus subject areas

  • Biochemistry


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