Development and utilization of 8-azidopurine nucleotide photoaffinity probes

The 8-azidopurine analogs of adenosine and guanine nucleotides have proved to be very useful probes for nucleotide-binding sites. In most systems they have proved to be effective mimics of the natural compounds with 1) both 8-azidoadenosine-3',5'-monophosphate and 8-azidoguanosine-3',5'-monophosphate activating their respective kinases, 2) 8-azidoguanosine-5'-triphosphate effecting tubulin polymerization and activation of adenylate cyclase, and 3) 8-azidoadenosine-5'-triphosphate appearing to be a substrate for a large number of ATPases and several kinases. As photoprobes they have been used to 1) isolate and study active site peptides; 2) determine the membrane sidedness and cellular location of binding sites; 3) detect the availability of various nucleotide-binding sites as cells progress through development, maturation, infectious stages, etc.; 4) study membrane-soluble partitioning of binding sites relative to nucleotide regulation of a biochemical process; 5) detect nucleotide-binding sites exposed by small molecules such as Ca2+ and calmodulin; and 6) detect potential catalytic and regulatory subunits of protein kinases found in preparations that actively phosphorylate endogenous substrates. The difference between the gamma-32P-labeled 8-azidopurine nucleotide triphosphate and the alpha-32P-labeled species has been used to study the in situ hydrolysis of the nucleotides on specific protein receptors and determine the fate of the produced nucleotide diphosphate. Such factors are important in studying the molecular dynamics of such systems as tubulin polymerization, G-actin to F-actin conversions, and GTP activation of adenylate cyclase. A review of techniques used and data obtained with these probes is presented.