Studies on the conversion of proinsulin to insulin. 3. Studies in vitro with a crude secretion granule fraction isolated from rat islets of Langerhans.

Proinsulin is converted to insulin in an impure secretion granule fraction prepared from rat islets of Langerhans that have been labeled before homogenization with [3H]leucine or [aH]arginine. During incubation of this particulate fraction at pH 6.3 and 37”, the initial rate of conversion of the endogenous labeled proinsulin is similar to that observed in whole islets, while externally added labeled proinsulin is not cleaved. Only intact granules catalyze conversion, and thus the pH optimum of about 6.0 for this process corresponds closely to that for granule stability. The most rapid in vitro cleavage of endogenous proinsulin is observed when the islets have been prelabeled with [3H]leucine for 30 min, followed by a 15-min “chase” to allow time for the transport of newly synthesized proinsulin to the Golgi apparatus and new secretory granules. Several proteinase inhibitors, including soybean trypsin inhibitor, pancreatic trypsin inhibitor, diisopropyl fluorophosphate, N-cr-p-tosyl-L-lysine chloromethyl ketone .HCl, benzamidine, P-nitrophenyl-P’-guanidinobenzoate .HCl, N-ethylmaleimide, and iodoacetate, do not inhibit conversion in vitro, possibly due to a lack of permeability of the granules to some of these substances; high concentrations of p-chloromercuribenzoate completely inhibit conversion. The products of in vitro conversion have been characterized by polyacrylamide gel and thin layer electrophoresis as rat insulins I and II and their corresponding C-peptides. The residual proinsulin fraction after incubation consists mainly of partly cleaved intermediate forms. When islets are prelabeled with [3H]arginine before preparation of the granule fraction, in vitro conversion is accompanied by the release from the cleavage regions of free arginine rather than dipeptides of arginine or lysylarginine. Granule preparations disrupted by repeated freeze-thawing lose their ability to introduce cleavages in intact proinsulin but are still able to rapidly remove COOH-terminal arginine residues from lightly trypsinized proinsulin. A low level of