Direct photoaffinity labeling of individual cytosolic domains of adenylyl cyclase by [P-32]2 '-deoxy-3 '-AMP and [alpha-P-32]5 '-ATP

The susceptibility of purines to form a covalent attachment with proteins u pon exposure to UV irradiation was applied to adenylyl cyclase by use of [P -32]2'-d-3'-AMP, a dead-end inhibitor that binds to the posttransition conf iguration of the enzyme. [P-32]2'-d-3'-AMP was synthesized enzymatically. I t and [c-P-32]5'-ATP were used for direct photocross-linking to individuall y expressed cytosolic domains of adenylyl cyclase, Both the C-1 domain of t he type V isozyme (VC1) and the C-2 domain of the type II isozyme (IIC2) we re labeled, whether alone or combined, upon photolysis of [P-32]2'-d-3'-AMP in the presence of acetone, Labeling of VC1 and IIC2 was greatly enhanced in the presence of PPi, was almost completely suppressed by 50 mu M 2',5'-d ideoxy-3'-ATP, the most potent reported P-site inhibitor of adenylyl cyclas es, but was partially suppressed by I mM 3'-IMP, a ligand that does not inh ibit the enzyme tia the P-site, Neither 3':5'-cAMP nor 5'-ATP had a major e ffect on labeling by [P-32]2'-d-3'-AMP. Direct cross-linking of VC1 with [a lpha-P-32]5'-ATP was substantially suppressed by 2',5'-dideoxy-3'-ATP and p artially suppressed by 2'-d-3'-AMP, whereas cross-linking of IIC2 was less affected by the 3'-triphosphate. The data imply that either cytosolic domai n can interact directly with either substrate or P-site ligand and that sub unit interaction modifies the susceptibility of each domain to UV-induced c ovalent modification by either [alpha-P-32]5'-ATP or [P-32]2'-d-3'-AMP.