Nucleoside phosphatase activities on pig pancreas zymogen granule membranes analyzed by non-denaturing polyacrylamide gel electrophoresis.
The membrane of the pancreatic zymogen granule plays an important part in the sequence of storage, transport and exocytosis of digestive enzymes. While much is known on stimulus-secretion coupling, very little is understood about how the storage organelles move in the cytoplasm to the luminal plasma membrane and why and how they fuse with it to release the contents. It is assumed that nucleoside phosphatases are involved in these energy consuming processes. Pancreatic zymogen granule membranes contain one major glycoprotein, GP-2, and a few minor proteins all with unknown functions. In order to identify functions we have purified zymogen granule membranes from pig pancreas, solubilized the proteins under non-denaturing conditions with the detergent CHAPS and characterized the extracted proteins by polyacrylamide gel electrophoresis, histochemistry and lectins. Three major protein bands, often fused in one broad band, revealed enzymatic activity for adenosine-, cytidine-, inositol- and guanidine- di- and triphosphates by the precipitation of liberated phosphate by Pb(NO3)2. This activity was sensitive to known ATP diphosphohydrolase inhibitors. The band with activity arises from a 92 kDa glycoprotein. A different narrow band showed monophosphatase activity for AMP, GMP, IMP and CMP. Some of the activities were inhibited by different lectins, indicating glycosyl groups near the active site. Electron microscopical cytochemistry confirmed a nucleoside phosphatase activity on granule membranes. Our results show for the first time that the nucleoside phosphatase activity of the zymogen granule membranes is carried by a 92 kDa glycoprotein, probably the known self-associating form of GP-2. The hydrolysis of tri- and diphosphate nucleotides could provide the energy required by exocytosis.
D000818:Animals; D002499:Centrifugation, Density Gradient; D002793:Cholic Acids; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000648:ultrastructure; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism* / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism; D009708:Nucleotidases / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology* / Q000378:metabolism; D013552:Swine
D002793:Cholic Acids; D003902:Detergents; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D008565:Membrane Proteins; D009711:Nucleotides; D009708:Nucleotidases; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate