8-azidoguanosine-triphosphate and 8-azidoadenosine-3--5--monophosphate

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.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Affinity Labels; Animals; Azides; Calcium; Cyclic AMP; Cyclic GMP; Guanosine Triphosphate; Nucleotides; Phosphorylation; Photochemistry; Receptors, Cyclic AMP

It has been clearly shown that the action of several hormones is differentially mediated intracellularly by nucleotides containing either adenosine or guanosine base units. To study the protein-nucleotide interactions involved in several complex biological systems our laboratory has synthesized several 8-azido-adenosine (8-N3 A) and 8-azidoguanosine (8-N3 G) derivatives of naturally occurring nucleotides. Modification of the nucleotides in the 8-position of the purine ring was done because: a) 8-substituted derivatives of cAMP and cGMP activated their respective protein kinases at physiological concentrations and were much less susceptible to hydrolysis by specific phosphodiesterases (PDE's) and b) substitution at the 8-position was much less likely to disturb the preferential and selective binding of adenosine versus guanosine nucleotides by enzymes that are specifically regulated by such interactions. This would allow studies of guanosine nucleotide specific binding in the presence of both adenosine nucleotides and adenosine nucleotide binding proteins, and vice-versa. In general, such has been the case and [32P] 8-N3 cAMP and [32P] 8-N3 cGMP have been used effectively to study their respectively activated protein kinases in several systems. Also, [32P] 8-N3 ATP has been used to study several ATPases and kinases while [gamma 32P] 8-N3 GTP has been shown effective for studies on tubulin and the G-regulatory protein (G/N) of adenylyl cyclase (A.C.). Several observations suggest that there must be important physical and energetic tie-ins between external hormone binding and the loading and unloading of specific internal nucleotide binding sites. These binding sites may be activator signals for protein kinases (e.g., cAMP protein kinase regulatory subunit), or cyclases (e.g., G/N proteins of A.C.) or catalytic sites involved in the production or hydrolysis of cyclic nucleotides. The thrust of this article is to detail the use of 8-azidopurine photoaffinity analogs of ATP, GTP, cAMP and cGMP as they may be used to study hormone-mediated events which may or may not involve cyclic nucleotides as a second messenger.

Topics: Adenosine Triphosphate; Adenylyl Cyclases; Affinity Labels; Animals; Azides; Binding Sites; Cyclic AMP; Guanosine Triphosphate; Hormones; In Vitro Techniques; Nucleotides; Photochemistry; Protein Kinases