Identification of the catalytic subunit of the ATP diphosphohydrolase by photoaffinity labeling of high-affinity ATP-binding sites of pancreatic zymogen granule membranes with 8-azido-[alpha-32P]ATP.
Photoaffinity labeling has been performed on pancreatic zymogen granule membranes using 8-azido-[alpha-32P]ATP (8-N3-ATP). Proteins of 92, 67, 53, and 35 kdaltons (kDa) were specifically labeled. ATP (100 microM) inhibited very strongly the labeling with 8-N3-ATP, while ADP was much less potent, AMP and cAMP being inefficient. The apparent constants for 8-N3-ATP binding were in the micromolar concentration range for the four labeled proteins. Without irradiation, 8-N3-ATP was a competitive inhibitor (Ki = 2.66 microM) for the hydrolysis of ATP by the ATP diphosphohydrolase. The optimal conditions for the photolabeling of the 92- and 53-kDa proteins were pH 6.0 in presence of divalent cations. On the other hand the 67- and 35-kDa proteins required an alkaline pH and the addition of EDTA in the photolabeling medium. No proteins could be labeled on intact zymogen granules, showing that all the high-affinity ATP-binding sites of the membrane were located at the interior of the granule. Both the 92- and 53-kDa glycoproteins could bind to concanavalin A-Sepharose and be extracted in the detergent phase in the Triton X-114 phase separation system. These latter properties are typical of integral membrane proteins. In addition, the 53-kDa labeled protein was sensitive to endo-beta-N-acetylglucosaminidase digestion. Photolabeling with 8-N3-ATP of two different preparations of purified ATP diphosphohydrolase also led to the labeling of a 53-kDa protein. Thus among the four proteins labeled with 8-N3-ATP on the pancreatic zymogen granule membrane, the 53-kDa integral membrane glycoprotein was shown to bear the catalytic site of the ATP diphosphohydrolase.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000345:Affinity Labels; D000818:Animals; D001081:Apyrase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D001386:Azides; D001665:Binding Sites; D003594:Cytoplasmic Granules / Q000201:enzymology*; D004792:Enzyme Precursors / Q000032:analysis* / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology*; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis*; D010766:Phosphorylation; D013379:Substrate Specificity; D013552:Swine
D000345:Affinity Labels; D001386:Azides; D004792:Enzyme Precursors; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D001081:Apyrase