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J Biol Chem, Vol. 273, Issue 49, 32416-32420, December 4, 1998

Direct Photoaffinity Labeling of Individual Cytosolic Domains of Adenylyl Cyclase by [³²P]2'-deoxy-3'-AMP and [-³²P]5'-ATP

Sergey Doronin, Carmen Dessauer^§, and Roger A. Johnson

From the  Department of Physiology and Biophysics, Health Sciences Center, State University of New York, Stony Brook, New York 11794-8661 and ^§ Department of Pharmacology University of Texas Southwestern Medical Center, Dallas, Texas 75235

The susceptibility of purines to form a covalent attachment with proteins upon exposure to UV irradiation was applied to adenylyl cyclase by use of [³²P]2'-d-3'-AMP, a dead-end inhibitor that binds to the post-transition configuration of the enzyme. [³²P]2'-d-3'-AMP was synthesized enzymatically. It and [-³²P]5'-ATP were used for direct photocross-linking to individually expressed cytosolic domains of adenylyl cyclase. Both the C₁ domain of the type V isozyme (VC₁) and the C₂ domain of the type II isozyme (IIC₂) were labeled, whether alone or combined, upon photolysis of [³²P]2'-d-3'-AMP in the presence of acetone. Labeling of VC₁ and IIC₂ was greatly enhanced in the presence of PP_i, was almost completely suppressed by 50 µM 2',5'-dideoxy-3'-ATP, the most potent reported P-site inhibitor of adenylyl cyclases, but was partially suppressed by 1 mM 3'-IMP, a ligand that does not inhibit the enzyme via the P-site. Neither 3':5'-cAMP nor 5'-ATP had a major effect on labeling by [³²P]2'-d-3'-AMP. Direct cross-linking of VC₁ with [-³²P]5'-ATP was substantially suppressed by 2',5'-dideoxy-3'-ATP and partially suppressed by 2'-d-3'-AMP, whereas cross-linking of IIC₂ was less affected by the 3'-triphosphate. The data imply that either cytosolic domain can interact directly with either substrate or P-site ligand and that subunit interaction modifies the susceptibility of each domain to UV-induced covalent modification by either [-³²P]5'-ATP or [³²P]2'-d-3'-AMP.

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