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

1986 ◽  
Vol 64 (5) ◽  
pp. 456-462 ◽  
Author(s):  
Jean Paquette ◽  
François A. Leblond ◽  
Marlyne Beattie ◽  
Denis LeBel
Keyword(s):  
Membrane Protein ◽  
Gel Filtration ◽  
Soluble Form ◽  
Major Protein ◽  
Zymogen Granule ◽  
Chloromethyl Ketone ◽  
Reducing Conditions ◽  
Immunochemical Quantitation ◽  
Granule Membrane ◽  
Different Levels

The major protein of the pig pancreatic zymogen granule membrane is an integral glycoprotein of 92 × 103 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-α-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. It is concluded that as much of the 92-kDa protein is soluble in the content as it is anchored in the membrane. The protease responsible for its degradation upon addition of DTT seems to be closely associated with the protein and could be involved in its posttranslational solubilization leading to its secretion.

Processing of the Major Pancreatic Zymogen Granule Membrane Protein, GP2

Pancreas ◽  
2002 ◽  
Vol 24 (4) ◽  
pp. 336-343 ◽  
Author(s):  
Benjamin A. Fritz ◽  
Clinton S. Poppel ◽  
Matthew W. Fei ◽  
Anson W. Lowe
Keyword(s):  
Membrane Protein ◽  
Zymogen Granule ◽  
Granule Membrane

scholarly journals Protection from Pancreatitis by the Zymogen Granule Membrane Protein Integral Membrane-associated Protein-1

2002 ◽  
Vol 277 (52) ◽  
pp. 50725-50733 ◽  
Author(s):  
Takuji Imamura ◽  
Minoru Asada ◽  
Sherri K. Vogt ◽  
David A. Rudnick ◽  
Mark E. Lowe ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Zymogen Granule ◽  
Granule Membrane

scholarly journals Distribution and partial purification of a liver membrane protein capable of inactivating cytosol enzymes

1980 ◽  
Vol 186 (2) ◽  
pp. 571-579 ◽  
Author(s):  
G L Francis ◽  
F J Ballard
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Major Protein ◽  
Major Protein Band ◽  
Glucose 6 Phosphate Dehydrogenase

1. The inactivation of cytosol enzymes in liver extracts was carried out by several subcellular fractions, with plasma membranes having the highest specific activity. Rough and smooth microsomal fractions were both active, whereas lysosmal inactivation capacity appeared to be derived entirely from contaminating plasma-membrane fragments. 2. Inactivation capacity in liver fractions was derived from parenchymal cells. Of the non-liver cells tested, plasma membranes from H35 hepatoma cells were able to inactivate glucose 6-phosphate dehydrogenase (EC 1.1.1.49), adipocyte “ghosts” showed slight activity and erythrocyte and reticulocyte “ghosts” were inactive. 3. Liposomes prepared from pure lipids with net negative, positive or neutral charge did not possess inactivation capacity. 4. Liver plasma-membrane inactivation capacity was destroyed by heating at 50 degrees C. 5. Inactivation factor solubilized from membranes by trypsin plus Triton X-100 treatment was partially purified by (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and hydroxyapatite chromatography. 6. Partially purified inactivation factor analysed by gel electrophoresis gave a major protein band that co-migrated with capacity for inactivation of glucose 6-phosphate dehydrogenase. 7. It is concluded that inactivation factor is a membrane protein whose intracellular distribution and other properties are consistent with a possible role for this activity in the initial step of protein degradation.

Structure of the pig pancreatic GP-2: role of intramolecular disulfides in the resistance to proteolysis

1989 ◽  
Vol 67 (6) ◽  
pp. 281-287 ◽  
Author(s):  
Denis Lebel ◽  
Jean Paquette
Keyword(s):  
Disulfide Bonds ◽  
Hydrophobic Interaction Chromatography ◽  
Zymogen Granule ◽  
Proteolytic Digestion ◽  
Tryptic Fragment ◽  
Reducing Conditions ◽  
Granule Membrane ◽  
Reconstituted System ◽  
Specific Degradation

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.Key words: GP-2, zymogen granule, disulfide, exocrine, pancreas, secretion.

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