Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles.
Proinsulin is a single polypeptide chain composed of the B and A subunits of insulin joined by the C-peptide region. Proinsulin is converted to insulin during the maturation of secretory vesicles by the action of two proteases and conversion is inhibited by ionophores that disrupted intracellular H+ gradients. To determine if conversion of prohormone to hormone actually occurs in an acidic secretory vesicle, cultured rat islet cells were incubated in the presence of 3-(2,4-dinitroanilino)-3' amino-N-methyldipropylamine (DAMP), a basic congener of dinitrophenol that concentrates in acidic compartments and is retained there after aldehyde fixation. The cells were processed for indirect protein A-gold colocalization of DAMP, using a monoclonal antibody to dinitrophenol, and proinsulin, using a monoclonal antibody that exclusively reacts with the prohormone. The average density of DAMP-specific gold particles in immature secretory vesicles that contained proinsulin was 71/micron 2 (18 times cytoplasmic background), which indicated that this compartment was acidic. However, the density of DAMP-specific gold particles in the insulin-rich mature secretory vesicle averaged 433/micron 2. This suggests that although proinsulin conversion occurs in an acidic compartment, the secretory vesicles become more acidic as they mature. Since the concentration of anti-proinsulin IgG binding in secretory vesicles is inversely proportional to the conversion of proinsulin to insulin, we were able to determine that maturing secretory vesicles had to reach a critical pH before proinsulin conversion occurred.
D000818:Animals; D000911:Antibodies, Monoclonal; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D007328:Insulin / Q000032:analysis / Q000235:genetics*; D007515:Islets of Langerhans / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D010447:Peptide Hydrolases / Q000378:metabolism*; D011384:Proinsulin / Q000032:analysis / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D051381:Rats; D011919:Rats, Inbred Strains
D000911:Antibodies, Monoclonal; D007328:Insulin; D011384:Proinsulin; D010447:Peptide Hydrolases