Structure of the pig pancreatic GP-2: role of intramolecular disulfides in the resistance to proteolysis.
GP-2 is the major membrane glycoprotein characteristic of the pancreatic zymogen granule membrane. When granules are lysed in the presence of DTT, GP-2 becomes completely and specifically degraded. This proteolysis was reproducible with the same characteristics in the purified granule membrane. The protease was purified from this source using hydrophobic interaction chromatography. The proteolytic activity was identified as a 29-kDa protein because, in a reconstituted system containing both the purified GP-2 and the 29-kDa protein, the proteolytic degradation of GP-2 was sensitive to the same spectrum and concentrations of inhibitors or reducing agents as in the membrane. The activity was characteristic of a serine protease. It was also shown that GP-2 only becomes sensitive to proteolytic digestion when its disulfide bonds are reduced, and that DTT does not activate the protease. Seven intramolecular disulfide bonds were identified on GP-2. All of them are located in a 65-kDa tryptic fragment that is very resistant to exogenous proteases under nonreducing conditions. Because of the quite specific degradation of GP-2 under reducing conditions, we believe that the 29-kDa protease must be closely associated with GP-2 on the membrane. This protease could be responsible, in part, for the solubilization of the GP-2 from the membrane into the zymogen granule content and its resulting secretion by the pancreas.
LeBel
D
D
Départment de Biologie, Faculté des Sciences, Université de Sherbrooke, Québec, Canada.
Paquette
J
J
D000478:Alkylation; D000818:Animals; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000032:analysis; D006868:Hydrolysis; D008562:Membrane Glycoproteins / Q000032:analysis*; D008970:Molecular Weight; D010179:Pancreas / Q000032:analysis*; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000032:analysis; D013552:Swine
D004220:Disulfides; D004792:Enzyme Precursors; D008562:Membrane Glycoproteins; D013438:Sulfhydryl Compounds; D010447:Peptide Hydrolases