Identification of a glycosylphosphatidylinositol-anchored glycoprotein with nucleoside phosphatase activity on the membrane of pig pancreatic zymogen granules.
The molecular events between the second messenger-mediated triggering of regulated exocytosis and the subsequent fusion of the secretory granules with the apical plasma membrane are unclear. The glycoprotein GP-2, the most abundant of the very few proteins of the pancreatic zymogen granule membrane has been cloned and sequenced in dog and rat, but no (enzymatic) function has so far been ascribed to it. Nucleoside phosphatase activities associated with the pig zymogen granule membrane were recently assumed to be related to GP-2. To identify the protein(s) carrying these activities we have used a novel combination of native and denaturing one- and two-dimensional polyacrylamide gel electrophoresis in the detergents CHAPS, Triton X-100 or SDS. Histochemical examination on the gels and incubation with lectins and phosphatidylinositol phospholipase-C have allowed characterization of the protein with the nucleoside di- and tri-phosphatase activities. SDS-PAGE of the single protein spot with nucleoside phosphatase activity excised from Triton X-100 2-dimensional gels showed the presence of 92 kDa and 67 kDa glycoproteins. The isolated protein had an isoelectric point of 5.2, formed high molecular weight complexes, was shown to be glycosylphosphatidylinositol-anchored and contained complex carbohydrate structures. It hydrolyses di- and tri-phosphate nucleotides in dependence of the glycosylphosphatidylinositol anchor and is sensitive to non-mitochondrial diphosphohydrolase inhibitors. In summary, this paper identifies GP-2 as a nucleoside phosphatase within the zymogen granule membrane, suggesting it may be involved in energy-requiring processes on the cytosolic side of the granules.
Soriani
M
M
Departement für Innere Medizin, Universitätsspital Zürich, Switzerland.
Freiburghaus
A U
AU
D000818:Animals; D003594:Cytoplasmic Granules / Q000648:ultrastructure; D004285:Dogs; D004792:Enzyme Precursors / Q000032:analysis*; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D007425:Intracellular Membranes / Q000201:enzymology*; D007797:Laminin / Q000032:analysis*; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology*; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis*; D051381:Rats; D013552:Swine
D004792:Enzyme Precursors; D017261:Glycosylphosphatidylinositols; D007797:Laminin; D010744:Phosphoric Monoester Hydrolases