Effect of pH on Conversion of Proinsulin to Insulin by a Subcellular Fraction of Rat Islets 1

Abstract

Previous studies on insulin biosynthesis in rats indicate that insulin is formed by way of a precursor (proinsulin) and one or more intermediates (1). A delay of 10–20 min. between proinsulin synthesis and onset of conversion has been demonstrated. Within this same period of time proinsulin is rapidly accumulated into the secretion granule containing fraction of the cell. The T_1/2 for conversion of proinsulin to insulin is approximately 1 hr and the newly synthesized insulin appears to be secreted after 1 hr (2).

Cell fractionation studies (3, 4) and electron microscopic radioautography indicate that proinsulin is synthesized on the rough endoplasmic reticulum and sequestered into the secretion granules presumably by way of the Golgi apparatus. These findings have suggested that the conversion of proinsulin to insulin occurs within the Golgi apparatus and/or the developing secretion granules. The distribution of labeled proinsulin and insulin in subcellular fractions of isolated rat islets is consistent with this hypothesis (4). Also several inhibitors of energy metabolism have demonstrated an inhibition of the conversion of proinsulin to insulin, when added prior to the presumed transport of the proinsulin from the endoplasmic reticulum to the Golgi region (5).

It has been suggested that the enzyme necessary for conversion of proinsulin to insulin is trypsin-like, possibly acting in conjunction with a protease having a specificity similar to carboxypeptidase B (6). Studies with thiol inhibitors indicate that the transforming enzyme may belong to a group of proteases having a thiol in the active center, such as cathepsin B. We report here studies on the proteolysis of endogenous and exogenous proinsulin as a function of pH and on the concentration of proteolytic activity in the secretion granule-containing fraction.

Methods and Materials. Islets were isolated from (350–400 g) male Holtzman rats by a modification of the method of Moskalewski (7) developed by Lacy and Kostianovsky (8). This method has previously been reported (9).