Shoshani I, Qui H, Johnson F, Taussig R, Johnson R A
Department of Physiology and Biophysics, Health Sciences Center, Stony Brook, NY 11794-8661, USA.
Biochim Biophys Acta. 1995 Aug 17;1245(1):37-42. doi: 10.1016/0304-4165(95)00069-n.
Azidoiodophenyl-analogs of 2',5'-dideoxyadenosine were synthesized and tested as potential 'P'-site selective affinity probes for adenylyl cyclases. The 3'-substituted analogs included: 1: 3'-[(4-nitrophenyl)-acetyl]-2',5'-dideoxy-adenosine 2: 3'-[(4-nitrophenyl)-butyryl]-2',5'-dideoxyadenosine 3: 3'-[(4-azido-3-iodophenyl)-acetyl]-2',5'-dideoxyadenosine and 4: 3'-[(4-azido-3-iodophenyl)-butyryl]-2',5'-dideoxyadenosine. The azidoiodo-phenyl-analogs inactivated adenylyl cyclase irreversibly and in a light-dependent manner. This was observed with detergent-dispersed enzyme from rat brain, purified native enzyme from bovine brain, and recombinant Type I bovine adenylyl cyclase expressed in membranes from fall army worm ovarian (Sf9) cells. Inactivation of the recombinant enzyme was inversely dependent on ATP concentration and was not completely prevented by 2',5'-dideoxyadenosine. Inhibition kinetics with the recombinant enzyme in the absence of light suggested two sites of inhibition, whereas with the native Type I enzyme inhibition kinetics exhibited a straightforward noncompetitive mechanism. Occupation of either or both sites by ligand protected the enzyme against denaturation by UV-irradiation per se. The data are consistent with inactivation of the recombinant enzyme occurring both through the 'P'-site and the catalytic active site, but suggest that this is a characteristic of the recombinant enzyme and is not dependent on the probes per se. The data suggest the potential for independent interactions of such ligands with different sites on a given enzyme and also with other enzymes containing adenosine or adenine nucleotide binding domains.
