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

Instituto de Investigació en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Apdo. postal 187, Guanajuato, Gto., 36000 Mexico

¹Author for correspondence: Guadalupe Martinez-Cadena. Tel: +52 473 24996. Fax: +52 473 24302. e-mail: margua@quijote.ugto.mx

ABSTRACT

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 pl 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 HgCI₂, 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 MgCI₂ and by 250 µM 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 MgCI₂, 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 MgCI₂. 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-labelling of the soluble fraction with [-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.

This article has been cited by other articles:

				M. Deveze-Alvarez, J. Garcia-Soto, and G. Martinez-Cadena Glyceraldehyde-3-phosphate dehydrogenase is negatively regulated by ADP-ribosylation in the fungus Phycomyces blakesleeanus Microbiology, September 1, 2001; 147(9): 2579 - 2584. [Abstract] [Full Text] [PDF]