pH-independent and -dependent cleavage of proinsulin in the same secretory vesicle

By quantitative immunoelectron microscopy and HPLC, we have studied the effect of disrupting pH gradients, by ammonium chloride, on proinsulin conversion in the insulin-producing B-cells of the islets of langerhans. Proinsulin content and pH in single secretory vesicles were measured on consecutive serial sections immunostained alternately with anti-proinsulin or anti-dinitrophenol (to reveal the pH-sensitive probe DAMP) antibodies. Radioactivity labeled proinsulin, proinsulin cleavage intermediates, and insulin were quantitated by HPLC analysis of extracts of islets treated in the same conditions. Cleavage at the C- peptide/A-chain junction is significantly less sensitive to pH gradient disruption than that of the B-chain/C-peptide junction, but the range of pH and proinsulin content in individual vesicles indicate that both cleavages occur in the same vesicle released from the TGN.

[1]  P. Halban,et al.  Slow cleavage at the proinsulin B-chain/connecting peptide junction associated with low levels of endoprotease PC1/3 in transformed beta cells. , 1993, The Journal of biological chemistry.

[2]  P. Halban,et al.  Kinetics of Proinsulin Conversion in Human Islets , 1993, Diabetes.

[3]  D. Steiner,et al.  The new enzymology of precursor processing endoproteases. , 1992, The Journal of biological chemistry.

[4]  J. Hutton,et al.  Kinetic analysis of the type-1 proinsulin endopeptidase by a monoclonal antibody-based immunoadsorbent assay. , 1992, The Biochemical journal.

[5]  S. Arden,et al.  Identification of the type 2 proinsulin processing endopeptidase as PC2, a member of the eukaryote subtilisin family. , 1992, The Journal of biological chemistry.

[6]  W. Lennarz,et al.  Proteolysis of the major yolk glycoproteins is regulated by acidification of the yolk platelets in sea urchin embryos , 1992, The Journal of cell biology.

[7]  P. Halban,et al.  Differential rates of conversion of rat proinsulins I and II. Evidence for slow cleavage at the B-chain/C-peptide junction of proinsulin II. , 1991, The Biochemical journal.

[8]  L. Villa-komaroff,et al.  Oxidation of rat insulin II, but not I, leads to anomalous elution profiles upon HPLC analysis of insulin‐related peptides , 1988, FEBS letters.

[9]  R. Mains,et al.  The role of a low pH intracellular compartment in the processing, storage, and secretion of ACTH and endorphin. , 1988, The Journal of biological chemistry.

[10]  H. Davidson,et al.  Intraorganellar calcium and pH control proinsulin cleavage in the pancreatic β cell via two distinct site-specific endopeptidases , 1988, Nature.

[11]  P. Halban,et al.  The intracellular handling of insulin-related peptides in isolated pancreatic islets. Evidence for differential rates of degradation of insulin and C-peptide. , 1988, The Biochemical journal.

[12]  D. L. Quinn,et al.  The trans-most cisternae of the Golgi complex: A compartment for sorting of secretory and plasma membrane proteins , 1987, Cell.

[13]  L. Orci,et al.  Use of a synthetic peptide antigen to generate antisera reactive with a proteolytic processing site in native human proinsulin: demonstration of cleavage within clathrin-coated (pro)secretory vesicles. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Richard G. W. Anderson,et al.  Proteolytic maturation of insulin is a post-Golgi event which occurs in acidifying clathrin-coated secretory vesicles , 1987, Cell.

[15]  L. Orci,et al.  Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles , 1986, The Journal of cell biology.

[16]  Q. Al-Awqati.  Proton-translocating ATPases. , 1986, Annual review of cell biology.

[17]  I Mellman,et al.  Acidification of the endocytic and exocytic pathways. , 1986, Annual review of biochemistry.

[18]  L. Orci,et al.  Direct identification of prohormone conversion site in insulin-secreting cells , 1985, Cell.

[19]  O. Madsen,et al.  Human Proinsulin-Specific Antigenic Determinants Identified by Monoclonal Antibodies , 1984, Diabetes.

[20]  M. Brown,et al.  Visualization of acidic organelles in intact cells by electron microscopy. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[21]  E. Kellenberger,et al.  Specimen preparation for electron microscopy using low temperature embedding resins , 1982, Journal of microscopy.

[22]  L Orci,et al.  Ultrastructural localization of intracellular antigens by the use of protein A-gold complex. , 1978, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[23]  M. Maxwell.  Two rapid and simple methods used for the removal of resins from 1.0 μm thick epoxy sections , 1978, Journal of microscopy.

[24]  P. Lacy,et al.  Method for the Isolation of Intact Islets of Langerhans from the Rat Pancreas , 1967, Diabetes.