Evidence for an arginine-specific mono(ADP-ribosyl)transferase in dormant spores of the fungus Phycomyces blakesleeanus

• Published in: Microbiology (Society for General Microbiology) 1996-10, Vol.142 (10), p.2907-2912
• Main Authors: Deveze-Alvarez, M, Garcia-Soto, J, Martinez-Cadena, G
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Microbiology; Mycology; plant biochemistry; plant physiology
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/13500872-142-10-2907

A soluble mono(ADP-ribosyl)transferase was detected in dormant spores of Phycomyces blakesleeanus. Soluble proteins incubated with [32P]NAD revealed, after two-dimensional electrophoresis, three major ADP-ribosylated substrates with molecular masses of 38, 37 and 36 kDa and pI values of 6.9, 8.1 and 4.6, respectively. When these endogenous substrates were first ADP-ribosylated with [32P]NAD and then incubated for different times with either 3 M hydroxylamine (pH 7.0) or 1 mM HgCl2, 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 by 10 mM MgCl2 and by 250 micromolar of the nitric-oxide-releasing agent sodium nitroprusside, and inhibited by 8 mM benzamide, 0.4 mM m-iodobenzylguanidine and 5 mM novobiocin. The three ADP-ribosylation inhibitors affected the germination of P. blakesleeanus spores, leaving them a swollen cells. The effect of MgCl2, GTP and ATP on the ADP-ribosylation of the endogenous proteins was studied. The presence of two additional [32P]ADP-ribosylated proteins of 57 and 55 kDa was observed in the absence of MgCl2. An increase in incorporation of radioactivity into the 55 kDa band was observed when the assay was performed 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-labeling of the soluble fraction with [alpha-32P]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.