title: Reducing conditions induce a total degradation of the major zymogen granule membrane protein in both its membranous and its soluble form. Immunochemical quantitation of the two forms.
abstract: The major protein of the pig pancreatic zymogen granule membrane is an integral glycoprotein of 92 X 10(3) daltons (Da) which amounts to 25% of the total proteins of this membrane. When zymogen granule membranes were prepared in presence of 5 mM dithiothreitol (DTT), this glycoprotein specifically vanished from the membrane preparation. During membrane purification two other fractions were produced out of the purified granules: a soluble fraction of zymogens referred to as granule content and a dense pellet. The possibility that DTT could release the 92-kDa protein from the membrane to these other fractions has been rejected. Altogether, addition of DTT during the lysis of the granules induced a total degradation of the 92-kDa protein. This hydrolysis could be inhibited by phenylmethylsulfonyl fluoride but not by N-alpha-p-tosyl-L-lysine chloromethyl ketone or L-1-tosylamide-2-phenylethylchloromethyl ketone. In the course of these experiments, using gel filtration of the granule content, it was found that the 92-kDa protein was also present in the granule content in the form of an aggregate of 300 kDa. A protease was present in this aggregate and could hydrolyse the 92-kDa protein upon addition of DTT. From immunoblotting studies and rocket immunoelectrophoresis, it was found that the soluble 92-kDa protein was antigenically similar to the membrane protein and that 44% of the immunoreactive glycoprotein of the granule was soluble in the content. A cross-reacting fragment of 65 kDa has been observed in all the fractions, yet at different levels.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000818:Animals; D002458:Cell Fractionation; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000648:ultrastructure; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera; D007122:Immunoelectrophoresis; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D010179:Pancreas / Q000378:metabolism* / Q000648:ultrastructure; D012995:Solubility; D013552:Swine
chemical_list: D007106:Immune Sera; D008565:Membrane Proteins; D004229:Dithiothreitol