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Molecular characterisation of a fungal mono(ADP-ribosyl)transferase
M Deveze-Alvarez1, J García-Soto, G Martínez-Cadena
1Instituto de Investigación en Biología Experimental Facultad de Química, Universidad de Guanajuato, México.
Abstract:
A soluble arginine-specific mono(ADP-ribosyl)transferase was detected in dormant spores of Phycomyces blakesleeanus. Soluble proteins incubated with [32P]NAD revealed, after a two dimensional electrophoretic separation, three major ADP-ribosylated substrates with molecular weights of 38, 37, and 36 kDa and pI values of 6.9, 8.1 and 4.6, respectively. The addition of MgCl2 stimulated the (ADP-ribosyl)transferase activity. This enzymatic activity was stimulated by 250 microM NO-releasing agent sodium nitroprusside and inhibited with 8 mM benzamide, 0.4 mM meta-IodoBenzylGuanidine (MIBG), and 0.5 mM novobiocin. The three ADP-ribosylation inhibitors affected the germination of Phycomyces spores. The concentrations necessary to inhibit 50% of the spore germination of Phycomyces were 0.05 mM, 0.2 mM, and 8 mM for novobiocin, MIBG, and benzamide, respectively. All the above inhibitors affected the germination process to the same extent, that is, they inhibited the tube protuberation, leaving the spores as swollen cells. These data suggest that ADP-ribosylation may be involved in the germination process of Phycomyces, particularly in germ-tube formation.
Insights
A soluble enzyme that modifies proteins was found in dormant Phycomyces blakesleeanus spores. This enzyme
Area of Science:
- Biochemistry
- Molecular Biology
- Mycology
Background:
- Dormant spores of Phycomyces blakesleeanus possess enzymatic machinery that is activated during germination.
- ADP-ribosylation is a post-translational modification with diverse cellular roles, but its function in fungal spore germination is not well understood.
Purpose of the Study:
- To investigate the presence and activity of ADP-ribosyltransferase in Phycomyces blakesleeanus dormant spores.
- To determine the potential role of ADP-ribosylation in the germination process of Phycomyces spores.
Main Methods:
- Detection and characterization of soluble arginine-specific mono(ADP-ribosyl)transferase activity in Phycomyces blakesleeanus dormant spores.
- Identification of ADP-ribosylated protein substrates using [32P]NAD and two-dimensional electrophoresis.
- Assay of enzyme activity modulation by MgCl2, sodium nitroprusside, benzamide, meta-IodoBenzylGuanidine (MIBG), and novobiocin.
- Evaluation of the effect of ADP-ribosylation inhibitors on Phycomyces spore germination and germ-tube formation.
Main Results:
- A soluble arginine-specific mono(ADP-ribosyl)transferase was identified in dormant Phycomyces blakesleeanus spores.
- Three major ADP-ribosylated substrates (38, 37, and 36 kDa) were detected.
- Enzyme activity was stimulated by MgCl2 and sodium nitroprusside, and inhibited by benzamide, MIBG, and novobiocin.
- Benzamide, MIBG, and novobiocin significantly inhibited Phycomyces spore germination, specifically blocking germ-tube protuberation.
Conclusions:
- ADP-ribosylation is present in dormant Phycomyces blakesleeanus spores.
- ADP-ribosylation plays a crucial role in the germination process of Phycomyces, particularly in germ-tube formation.