EVIDENCE FOR AN ARGININE-SPECIFIC MONO(ADP-RIBOSYL)TRANSFERASE IN DORMANT SPORES OF THE FUNGUS PHYCOMYCES-BLAKESLEEANUS

A soluble mono(ADP-ribosyl)transferase was detected in dormant spores of Phycomyces blakesleeanus. Soluble proteins incubated with [P-32]NAD revealed, after two-dimensional electrophoresis, three major ADP-ribo sylated substrates with molecular masses of 38, 37 and 36 kDa and pl v alues of 6 . 9, 8 . 1 and 4 . 6, respectively When these endogenous su bstrates were first ADP-ribosylated with [P-32]NAD and then incubated for different times with either 3 M hydroxylamine (pH 7 . 0) or 1 mM H gCl2 only hydroxylamine released the incorporated radioactivity after 30 min incubation. Additionally, agmatine was used as a substrate for this enzyme. These data suggest that the mono(ADP-ribosyl)transferase is an arginine-specific enzyme. This enzymic activity was stimulated b y 10 mM MgCl2 and by 250 mu M of the nitric-oxide-releasing agent sodi um nitroprusside, and inhibited by 8 mM benzamide, 0 . 4 mM m-iodobenz ylguanidine and 0 . 5 mM novobiocin. The three ADP-ribosylation inhibi tors affected the germination of P. blakesleeanus spores, leaving them as swollen cells. The effect of MgCl2, GTP and ATP on the ADP-ribosyl ation of the endogenous proteins was studied. The presence of two addi tional [P-32]ADP-ribosylated proteins of 57 and 55 kDa was observed in the absence of MgCl2. An increase in incorporation of radioactivity i nto the 55 kDa band was observed when the assay was performed in the p resence of GTP or ATE. The addition of Mg2+ together with either or bo th nucleotides eliminated the appearance of the 57 and 55 kDa bands, b ut intensified the 37 kDa band. Photoaffinity-labelling of the soluble fraction with [alpha-P-32]GTP revealed a 55 kDa band together with ot her proteins of 32 and 17 kDa. These results suggest that among the fi ve different endogenous substrates for the fungal mono(ADP-ribosyl)tra nsferase, the 55 kDa protein may be a GTP-binding protein.