P. Hoyer, James R. Owens, B. Haley
Jun 1, 1980
Citations
0
Influential Citations
29
Citations
Journal
Annals of the New York Academy of Sciences
Abstract
The purpose of this paper is to describe results, techniques, problems, and new experimental approaches that involve the use of nucleotide photoaffinity probes. It is our contention that photoprobes such as 8-azidoadenosine-3’,5’-cyclic monophosphate (8-N,cAMP), 8-azidoguanosine-3’,5’-cyclic monophosphate (8-N3cGMP), 8-azidoadenosine-5’-triphosphate (8-N,ATP), 8-azidoguanosine-5’-triphosphate (8-N3GTP) and will be extremely useful in determining the mechanisms by which their natural analogs regulate biological phenomena. Regulation by such nucleotides can be effected either by conformational changes caused by binding to a receptor molecule or by phosphorylation of a substrate molecule. Both of these situations require that the nucleotide in question occupy a specific site on a specific macromolucule. It is the determination of the extent of interaction between the nucleotide and the receptor protein that is of prime importance, as well as how, when, and where such interactions occur. Research in our laboratory has been directed a t the mechanisms through which cAMP and ATP regulate cellular events and certain related biochemical reactions. Our approach has been to use the 8-azido derivatives of both of these compounds; our earlier emphasis was on CAMP. Use of [32P]8-N3~AMP has shown that it is specific for cAMP binding sites and efficiently photoincorporated into receptor molecules under optimal conditions.’” The photolysis of 8-N3cAMP probably generates a nitrene that has the ability to react with any amino acid residue within the active site (FIGURE 1). Also, 8-N,cAMP is a good biological mimic of cAMP with regard to activating Type I and I1 protein kinases.’.’ It is not a good substrate for most phosphodiesterases (PDE), showing no significant hydrolysis with mammalian PDEs, but it is hydrolyzed by D. discoideum PDE?5 Previous results obtained using [32P]8-N3~AMP have shown that it may be used to determine several kinetic and physical properties of cAMP receptor molecules, such as approximate molecular weights and ligand binding affinities.’” It has been used to determine the cellular location of cAMP receptor proteins and when certain receptor proteins appear during a cell or life ~ y c l e . ~ . ~ It has also been used to determine the effects of cAMP on the membrane binding properties of Type I-M protein kinases’ regulatory (R,) and catalytic (C) subunits. These results indicate that this protein kinase is attached to the membrane through the R, subunit and that cAMP causes release of the C subunit, although the R,-CAMP complex remains bound to the