Direct photoaffinity labeling of proteins with adenosine 3'-[32P]phosphate 5'-phosphosulfate. Atractyloside inhibits labeling of a Mr = 34,000 protein in an adrenal medullary Golgi fraction.
Direct photoaffinity labeling with radioactively labeled adenosine 3'-phosphate 5'-phosphosulfate (PAPS) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography was used to identify PAPS binding proteins in a Golgi membrane preparation of bovine adrenal medulla. [3'-32P]PAPS was synthesized from adenosine 5'-phosphosulfate (APS) and [gamma-32P]ATP using APS kinase prepared from yeast and was purified by reverse-phase ion pair high performance liquid chromatography. Upon irradiation with UV light, [3'-32P]PAPS, as well as [35S]PAPS under conditions which minimized sulfotransferase-catalyzed incorporation of 35SO4 from [35S]PAPS into proteins, bound selectively to a 34-kDa protein of the Golgi membrane preparation. PAPS binding to the 34-kDa protein was strongly inhibited by the presence of 50 microM atractyloside. The 34-kDa PAPS binding protein therefore appears to be similar to the mitochondrial ATP/ADP translocator with regard to both molecular weight and inhibition by atractyloside of adenine nucleotide binding. Photoaffinity labeling will be useful in the purification and functional identification of the 34-kDa protein.
D000227:Adenine Nucleotides / Q000378:metabolism*; D000313:Adrenal Medulla / Q000378:metabolism*; D000818:Animals; D001278:Atractyloside / Q000494:pharmacology*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006027:Glycosides / Q000494:pharmacology*; D006056:Golgi Apparatus / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010777:Photochemistry
D000227:Adenine Nucleotides; D006027:Glycosides; D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; D001278:Atractyloside; D010724:Phosphoadenosine Phosphosulfate