title: Proteolytic conversion of proinsulin into insulin. Identification of a Ca2+-dependent acidic endopeptidase in isolated insulin-secretory granules.
abstract: The nature of the endoproteolytic activity involved in the post-translational processing of proinsulin has been investigated in rat insulinoma tissue. 125I-proinsulin was converted by lysed insulin-secretory granules into insulin via an intermediate form identified as des-dibasic-proinsulin. This activity co-localized with immunoreactive (endogenous) insulin and carboxypeptidase H upon subcellular fractionation of the tissue, indicating a secretory-granular location. Under optimized conditions, conversion was quantitative. Inhibitor studies demonstrated that processing occurred by a reaction sequence involving cleavage on the C-terminal side of the pairs of basic amino acids, with subsequent removal of the newly exposed basic residues by carboxypeptidase H. Endoproteolytic activity was abolished by EDTA and CDTA (1,2-cyclohexanediaminetetra-acetic acid), but not by 1,10-phenanthroline or by group-specific inhibitors of serine, thiol or acidic proteinases. Inhibition by EDTA and CDTA could be reversed by both Ca2+ and Zn2+, although the former appeared to be the ion of physiological importance. Addition of Ca2+ in the absence of chelators stimulated endoproteinase activity, with a maximal effect at 5 mM, a concentration consistent with the intragranular environment. Similarly the pH optimum of 5.5 coincides with the prevailing intragranular pH. Together these properties suggest that the Ca2+-dependent endopeptidase described here is involved in vivo in the proteolytic processing of proinsulin.
authors.affiliation: Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, U.K.
authors.forename: H W
authors.initials: HW
authors.lastname: Davidson
authors.forename: M
authors.initials: M
authors.lastname: Peshavaria
authors.forename: J C
authors.initials: JC
authors.lastname: Hutton
mesh_terms: D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D003594:Cytoplasmic Granules / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma / Q000378:metabolism; D011384:Proinsulin / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D007328:Insulin; D011480:Protease Inhibitors; D011384:Proinsulin; D010450:Endopeptidases; C054196:proinsulin endopeptidase I; D002118:Calcium