Agmatine toxin

Two soluble ARFs, sARFI and sARFII (Tsai et al., 1988), as well as a membrane-bound ARF (Tsai et al., 1987), were purified from bovine brain. sARFI and sARFII were later identified as ARFl and ARF3, respectively (Tsai et al., 1992). In the presence of cholate, ARF-stimulated ADP-ribosylation of Gs by cholera toxin was enhanced by DMPC (Tsai etal., 1988). However, DMPC/cholate was not required for ARF-stimulated auto-ADP-ribosylation of CTA (Tsai et al., 1988). sARFII-stimulated NAD agmatine ADP-ribosyltransferase activity was enhanced by SDS in a concentration-dependent manner 0.003 % produced maximal activity (Noda etal., 1990). 

Fig. 2. (right) ADP-ribosylation of a specific protein in bovine adrenal membranes by type D botulinum toxin. Membranes of bovine adrenal gland (100 pg) were incubated with 20 pg of type D botulinum neurotoxin and [ P]NAD at 30°C for 45 min, acid-precipitated, and subjected to SDS-polyacrylamide gel electrophoresis. Lane 1, control without the toxin lane 2, with the toxin lane 3, with the toxin plus 10 mM dithiothreitol lane 4, with the toxin plus 25 mM agmatine lane 5, with the toxin plus 25 mM L-arginine methyl ester lane 6, with 20 pg of cholera toxin. Reproduced from ref 5. 

To test the ability of ARF to enhance the ADP-ribosylation of simple guanidino compounds, its activity was examined in the NADragmatine ADP-ribosyltransferase assay (Table 1). ARF increased the toxin-catalyzed ADP-ribosylation of agmatine approximately 3-fold. As noted with the auto-ADP-ribosylation reaction, activation of ADP-ribosylagmatine formation was dependent on GTP or its non-hydrolyzable analogues. GDP, GDPpS, ATP and App(NH)p were consistently less effective. ARF also increased the toxin-catalyzed ADP-ribosylation of proteins unrelated to the... 

To determine the possible mechanism for stimulation of toxin by ARF, NAD agmatine ADP-ribosyltransferase activity was examined at different substrate concentrations. The principal effect of ARF was to decrease the Km s for both NAD and agmatine, with little change in the Vmax. Thus, at subsaturating substrate concentrations, as might be expected to occur in vivo, ARF would enhance the rate of ADP-ribosylation. 

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