Agmatine Assay

It is sometimes necessary to modify the conditions of the assays described here. In one specific example, conditions of the agmatine assay were changed to allow the assay of a second enzyme, phospholipase D (PLD), under identical conditions (Massenburg et al., 1994). With the identification of ARF as an activator of PLD (Brown et al., 1993 Cockcroft et al., 1994), it was desired to determine which mammalian ARFs activated PLD, and to identify ARF domains that were involved in the activation of CT and PLD. It was first determined that each ARF sample was active in Assay 2 in the presence of cardi-olipin each ARF was then assayed tor its ability to stimulate PLD in a separate assay (Massenburg et al., 1994). To compare the activities of ARF proteins in the CT and PLD assays, we used conditions under which both enzymatic activities could be measured. With the buffer conditions and sonified lipid vesicles of the composition required tor the pH-choline]-release assay used to measure PLD activity, all reagents required tor Assay 2 were added to create a dual assay system to determine the formation of ADP-ribosylagmatine determined as described above. To perform the PLD assay, radiolabeled vesicles of the same composition replaced the non-radiolabeled vesicles and choline release was determined. Although these conditions resulted in a lower ARF stimulation of CT activity than observed in Assay 2, they allowed direct comparison of ARF activation of CT and PLD. 

Fig. 1. Effect of agmatine and NAD on the activity of NAD arginine ADP-ribosyltransferase plotted with NAD as the variable substrate at different fixed concentrations of agmatine. Assays contained 50 mM potassium phosphate (pH 7.0), 1 mg ml ovalbumin, and the following concentrations of agmatine o 0.5 mAf 1 mAf 2 mM O 3 mAf. Reactions were performed at 30 C and initiated with erythrocyte transferase A (9.1 ng/assay). Inset Double reciprocal plot of the activity of transferase A in the absence of acceptor. Solid lines are the theoretical fit of the data to the random rapid equilibrium sequential mechanism described in the text...

Solutions are prepared in water using the reagents of the highest quality available, unless otherwise stated. They are divided into portions sufficient for 50 assays and stored at -20°C until use. Precipitates in agmatine, DMPC/cholate, and cardiolipin (formed with freezing) can be dissolved by heating to 50°C and vortex mixing. 

Table 2. Components of Assay 2 NAD agmatine ADP-ribosyltransferase Activity...

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... 

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