Differences in the nature of the interaction of insulin and proinsulin with zinc

Abstract

1. The reversible interaction of zinc with pig insulin and proinsulin has been studied at pH7 by equilibrium dialysis (ultrafiltration) and by sedimentation equilibrium and velocity measurements in the ultracentrifuge. Binding values calculated from equilibria, where the ratio of free to bound zinc was varied in the range 0.01:1-10:1, indicated that proinsulin and insulin each contained two main orders of zinc binding with very different affinities for the metal. 2. In equilibria containing low concentrations of free zinc (free: bound ratios of 0.01-0.1:1) both insulin and proinsulin aggregated to form soluble hexamers containing firmly bound zinc (up to 0.284g-atom/monomer) with an apparent intrinsic association constant of 1.9x10(6)m(-1). 3. Higher concentrations of zinc (free: bound ratios of 0.1-10.0:1) resulted in a progressive difference in the zinc binding, aggregation and solubility properties of the metal complexes of insulin and proinsulin. At the highest concentration of free zinc, proinsulin bound a total of more than 5.0g-atom/monomer and aggregated to form a mixture of soluble polymers (mainly 5.1S). In contrast, insulin bound a total of only 1.0g-atom/monomer and was almost completely precipitated from solution. 4. These results would indicate that the presence of the peptide segment connecting the insulin moiety in proinsulin does not prevent the firm binding of zinc to the insulin moiety and the formation of hexamers of zinc-proinsulin. At the same time although the connecting peptide contains additional sites of lower affinity for zinc, which should facilitate inter- and intra-molecular cross-linking, the general conformation of the zinc-proinsulin hexamer must preclude the formation of very large and close-packed aggregates that are insoluble in solutions at equilibrium.

Partial diversion of a mutant proinsulin (B10 aspartic acid) from the regulated to the constitutive secretory pathway in transfected AtT-20 cells.

“…It has recently been shown by immunocytochemistry in both AtT-20 cells (30) and pancreatic B cells (31) that secretory peptides condense in the trans-Golgi prior to secretory granule budding. Thus, it appears very likely that the self-association of proinsulin hexamers (32), in proximity to a presumed secretory granule targeting apparatus, results in the morphologically apparent local concentration of prohormone destined for secretory granules. Our findings are consistent with such a mechanism-namely, that monomeric (or dimeric) proinsulin can be targeted to granules, but at a much lower efficiency than the aggregated hexameric form.…”

Metal ions affect insulin-degrading enzyme activity

“…Metal-induced insulin oligomerization is an important and widely investigated issue and several studies dealing with the effect of metal ions on insulin conformation have already been published [62]. Indeed, information regarding IDE enzymatic digestions of the full length insulin in the presence of metal ions is very problematic due to insulin aggregation and to the different peptide conformations existing at different peptide/metal ratios [63,64].…”

Behavior in the Eukaryotic Secretory Pathway of Insulin-containing Fusion Proteins and Single-chain Insulins Bearing Various B-chain Mutations

“…Failure to secrete the single-chain insulin moiety cannot be explained by ICFP endoproteolysis within a "dead-end" compartment from which molecules can no longer be secreted, because simple deletion of the yeast KEX2 gene not only prevents ICFP processing but also increases the quantity of ICFP secreted. It is known that by liberating insulin from its precursor (in either mammalian cells or yeast), cleavage promotes a change in the biophysical properties of the processed product (5)(6)(7)(8)(9)(10)(11), and this might play a role in protein sorting in the lumen of the distal secretory pathway (12,13).…”