Direct photoaffinity labeling of individual cytosolic domains of adenylyl cyclase by [32P]2'-deoxy-3'-AMP and [alpha-32P]5'-ATP.
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 [alpha-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 PPi, was almost completely suppressed by 50 microM 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 [alpha-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 [alpha-32P]5'-ATP or [32P]2'-d-3'-AMP.
Doronin
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S
Department of Physiology and Biophysics, Health Sciences Center, State University of New York, Stony Brook, New York 11794-8661, USA.
Dessauer
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C
Johnson
R A
RA
D000096:Acetone / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000262:Adenylyl Cyclases / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology*; D003838:Deoxyadenine Nucleotides / Q000737:chemistry*; D011756:Diphosphates / Q000737:chemistry; D010761:Phosphorus Radioisotopes; D019920:Photoaffinity Labels; D010777:Photochemistry
D003838:Deoxyadenine Nucleotides; D011756:Diphosphates; D010761:Phosphorus Radioisotopes; D019920:Photoaffinity Labels; D000096:Acetone; C028191:2'-deoxy-3'-adenosine monophosphate; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases