Microbiology (1 996), 142, 2907-291 2 Printed in Great Britain Evidence for an arginine-specif ic mono(ADP- ribosy1)transferase in dormant spores of the fungus Phycomyces blakesleeanus Martha Deveze-Alvarez, Jesus Garcia-Soto and G uada I u pe Mart i nez-Cadena Author for correspondence : Guadalupe Martinez-Cadena. Tel : + 52 473 24996. Fax : + 52 473 24302. e-mail: margua@quijote.ugto.mx lnstituto de lnvestigacidn en B iolog ia Ex per i menta I, Facultad de Quimica, Universidad de Guanajuato, Apdo. postal 187, Guanajuato, Gto., 36000 Mexico A soluble mono(ADP-ribosy1)transferase was detected in dormant spores of Phycomyces blakesleeanus. Soluble proteins incubated with [',PINAD revealed, after two-dimensional electrophoresis, three major ADP-ribosylated substrates with molecular masses of 38,37 and 36 kDa and pl values of 609,801 and 46, respectively. When these endogenous substrates were first ADP-ri bosylated with [32P]NAD and then incubated for different times with either 3 M hydroxylamine (pH 7.0) or 1 mM HgCI,, 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- ribosy1)transferase is an arginine-specific enzyme. This enzymic activity was stimulated by 10 mM MgCI, and by 250 pM of the nitric-oxide-releasing agent sodium nitroprusside, and inhibited by 8 mM benzamide, 0.4 mM m- iodobenzylguanidine and 0.5 mM novobiocin. The three ADP-ribosylation inhibitors affected the germination of P. blakesleeanus spores, leaving them as swollen cells. The effect of MgCI,, GTP and ATP on the ADP-ribosylation of the endogenous proteins was studied. The presence of two additional [',P]ADP- ribosylated proteins of 57 and 55 kDa was observed in the absence of MgCI,. An increase in incorporation of radioactivity into the 55 kDa band was observed when the assay was pedormed in the presence of GTP or ATP. The addition of Mg2+ together with either or both nucleotides eliminated the appearance of the 57 and 55 kDa bands, but intensified the 37 kDa band. Photoaffinity-labelling of the soluble fraction with [E-~~P]GTP revealed a 55 kDa band together with other proteins of 32 and 17 kDa. These results suggest that among the five different endogenous substrates for the fungal mono(ADP- ribosyl)transferase, the 55 kDa protein may be a GTP-binding protein. Keywords : ADP-ribos yltransferase, Phycomyces blakesleeams, GTP-binding protein, dormant spores INTRODUCTION Mono-ADP-ribosylation is a post-translational modifi- cation of proteins by the enzymic transfer of ADP-ribose from NAD, resulting in alteration of the functional ribosyl)transferases have been identified in a range Of turkey red blood cells, hen and frog liver, and rabbit skeletal muscle. All of these enzymes catalyse the transfer .......................................................................................................................................................... Abbreviations: MIBG, rn-iodobenzylguanidine; NO, nitric oxide. of ADP-ribose from NAD to arginine (Williamson & Moss, 1990). Also, a cysteine-specific ADP-ribosyl- transferase has been purified from human erythrocytes (Jacobson et d., 1990). ADP-ribosylation is a reversible modification cycle of cysteine hydrolases have been identified in animal tissues (Moss etd., 1985; Williamson & Moss, 1990). The role of mono-ADP ribosylation in eukaryotes is less well charac- terized than in prokaryotes, although it has been sug- gested by different authors that this modification could Properties Of the Proteins* 'fono(ADP- proteins, since ADP-ribosylarginine and ADP-ribosyl- eukarYotic and tissues* have been Purified from in which the respective transferases have been detected 0002-0756 0 1996 SGM 2907