CHARACTERIZATION OF CHEMICAL INHIBITORS OF BREFELDIN A-ACTIVATED MONO-ADP-RIBOSYLATION

Brefeldin A, a toxin inhibitor of vesicular traffic, induces the selec tive mono-ADP-ribosylation of two cytosolic proteins, glyceraldehyde 3 -phosphate dehydrogenase and the novel GTP-binding protein BARS-50, He re, we have used a new quantitative assay for the characterization of this reaction and the development of specific pharmacological inhibito rs, Mono-ADP-ribosylation is activated by brefeldin A with an EC50 of 17.0 +/- 3.1 mu g/ml, but not by biologically inactive analogs includi ng a brefeldin A stereoisomer, Brefeldin A acts by increasing the V-ma x of the reaction, whereas it does not influence the K-m of the enzyme for NAD(+) (154 +/- 13 mu M). The enzyme is an integral membrane prot ein present in most tissues and is modulated by Zn2+, Cu2+, ATP (but n ot by other nucleotides), pH, temperature, and ionic strength, To iden tify inhibitors of the reaction, a large number of drugs previously te sted as blockers of bacterial ADP-ribosyltransferases were screened, T wo classes of molecules, one belonging to the coumarin group (dicumaro l, coumermycin A(1), and novobiocin) and the other to the quinone grou p (ilimaquinone, benzoquinone, and naphthoquinone), rather potently an d specifically inhibited brefeldin A dependent mono-ADP-ribosylation. When tested in living cells, these molecules antagonized the tubular r eticular redistribution of the Golgi complex caused by brefeldin A at concentrations similar to those active in the mono-ADP-ribosylation as say in vitro, suggesting a role for mono-ADP-ribosylation in the cellu lar actions of brefeldin A.