Specific interactions of pancreatic amylase at acidic pH. Amylase and the major protein of the zymogen granule membrane (GP-2) bind to immobilized or polymerized amylase.
Regulated secretory proteins are thought to be sorted in the trans-Golgi network towards the secretory granule via acidic aggregation. In the exocrine pancreas, amylase is one of the major zymogens. It is a basic protein of pI 8.6 and does not precipitate in acidic conditions. To identify the mechanism by which amylase aggregates in the acidic cisternae of the pancreatic trans-Golgi network, we have developed an in vitro model in which amylase was fixed to plastic microtiter plates. The fixed amylase was probed with two ligands: amylase itself and GP-2, the major protein of the zymogen granule membrane. Biotinylated amylase bound to fixed amylase in a strict pH-dependent manner with optimal binding between pH 5.0 and 5.7. The affinity of binding was in the nanogram range (Kd approximately 20.0 ng/mL) at pH 5.5. Acid binding of amylase was not reversible by incubation at neutral pH, nor could it be displaced by native amylase. GP-2 binding to fixed amylase was also pH dependent with optimal binding between pH 5.0 and 5.7. As for amylase, it was not reversible by incubation at neutral pH. GP-2 binding sites on fixed amylase appeared to be different from those of biotinylated amylase. While native and biotinylated amylase did not bind to GP-2, polymerized amylase precipitated GP-2 at acidic pH. Taken together these data suggest that slight modifications are sufficient to reveal on the amylase molecule binding sites for GP-2 and for amylase itself. These new binding capacities acquired at acidic pH could be involved in the cascade of reactions that lead to the in vivo formation of the immature secretory granule.
Jacob
M
M
Centre de recherche sur les mécanismes de sécrétion, Faculté des sciences, Université de Sherbrooke, Québec, Canada.
Lainé
J
J
LeBel
D
D
D000681:Amylases / Q000378:metabolism*; D000818:Animals; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism*; D004800:Enzymes, Immobilized; D058851:GPI-Linked Proteins; D006863:Hydrogen-Ion Concentration; D008562:Membrane Glycoproteins / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology*; D011108:Polymers; D011485:Protein Binding; D051381:Rats; D013552:Swine
D004792:Enzyme Precursors; D004800:Enzymes, Immobilized; D058851:GPI-Linked Proteins; C066161:Gp2 protein, rat; D008562:Membrane Glycoproteins; D011108:Polymers; D000681:Amylases