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.
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)
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
D007106:Immune Sera; D008565:Membrane Proteins; D004229:Dithiothreitol