ADPribosylation factor (ARF) and ras p21 use 8N3GTP as a mimic of GTP. 8N3GTP photoinserts effectively into each may be further modified on the gamma-phosphate with an extendable photoactive benzophenone (BP) group and still compete at the GTP site. ARF is a GTP binding protein that activates some bacterial toxins which use NAD+ to ADPribosylate other GTP binding proteins. Using [32P]8N3GTP and [32P]2N3NAD+ data has been obtained which shows that ARF alone contains both GTP and NAD+ binding domains. Further, the addition of ARF decreases by 50 percent the [32P]2N3NAD+ photoinsertion into interactive toxin with 50 percent of the labeling now appearing on ARF. This indicates a shared NAD+ site between ARF and toxin in the complex and has both structural and mechanistic implications. Therefore, the first specific aim will be to identify the NAD+ and GTP binding domains of ARF using [32P]8N3GTP and [32P]2N3NAD+ and to correlate photoinsertion with modification of biological activity. The second specific aim will be to synthesize and use NAD+ and GTP bifunctional photoprobes (azide plus benzophenone modified) to develop procedures to ~site specifically~ crosslink ARF with interactive toxin(s) through the GTP or NAD+ binding site(s). The ARF-toxin complex is being used as a model system to develop a general procedure which may be used to identify unknown proteins which interact with other G-regulatory proteins. The third specific aim will be to test the bifunctional GTP probes as biological mimics of GTP using ras p21 to develop procedures for identifying p21 ~nearest neighbor~ proteins. While many G-proteins of have been identified, the ~nearest neighbor~ interactive proteins they bind to are usually not known. The first aspect will be to crosslink p21 with known interactive proteins (GAP, Raf, etc) and this will be followed by studies to detect similar proteins in Xenopus homogenates. The major long term objective of this research is to develop a general procedure for the identification of the interactive proteins which bind different G-regulatory proteins, such as p21 and ARF.
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