Anglerfish proinsulin and insulin were selectively labeled with [14C]isoleucine, while proglucagon, conversion intermediate(s), and glucagon were selectively labeled with [aH]tryptophan. After various periods of continuous or pulse-chase incubation, islet tissue was subjected to subcellular fractionation. Fraction extracts were analyzed by gel filtration for their content of precursor, conversion intermediate(s), and product peptides. Of the seven subcellular fractions prepared after each incubation, only the microsome and secretory granule fractions yielded significant amounts of labeled insulin-related and glucagon-related peptides. After short-pulse incubations, levels of both [14C]proinsulin and [3H]proglucagon (mol wt -12,000) were highest in the microsome fraction. This fraction is therefore identified as the site of synthesis. With increasing duration of continuous incubation or during chase incubation in the absence of isotopes, proinsulin, proglucagon, and conversion intermediate(s) are transported to secretory granules. Conversion of proinsulin to insulin and proglucagon to a -4,900 mol wt conversion intermediate and 3,500 mol wt glucagon occurs in the secretory granules. Converting activity also was observed in the microsome fraction. The recovery of most of the incorporated radioactivity in microsome and secretory granule fractions indicates that the newly synthesized islet peptides are relegated to a membranebound state soon after synthesis at the RER is completed. This finding supports the concept of intracisternal sequestration and intragranular maintenance of peptides synthesized for export from the cell of origin.Results from recent studies designed to ascertain the mode of biosynthesis of glucagon indicate that a biosynthetic precursor is involved (8,(27)(28)(29)(30)(31)(32)(33)47). Evidence from other studies performed over the last 10 yr indicates that many, if not all, peptide hormones are synthesized as peptides larger than the biologically active circulating hormone and are cleaved enzymatically before secre-

Anglerfish islets were homogenized in 0.25 M sucrose and separated into seven separate subcellular fractions by differential and discontinuous density gradient centrifugation. The objective was to isolate microsomes and secretory granules in a highly purified state. The fractions were characterized by electron microscopy and chemical analyses. Each fraction was assayed for its content of protein, RNA, DNA, immunoreactive insulin (IRI), and immunoreactive glucagon (IRG). Ultrastructural examination showed that two of the seven subcellular fractions contain primarily mitochondria, and that two others consist almost exclusively of secretory granules. A fifth fraction contains rough and smooth microsomal vesicles. The remaining two fractions are the cell supernate and the nuclei and cell debris. The content of DNA and RNA in all fractions is consistent with the observed ultrastructure. More than 82% of the total cellular IRI and 89% of the total cellular IRG are found in the fractions of secretory granules. The combined fractions of secretory granules and microsomes consistently yield >93% of the total IRG. These results indicate that the fractionation procedure employed yields fractions of microsomes and secretory granules that contain nearly all the immunoassayable insulin and glucagon found in whole islet tissue. These fractions are thus considered suitable for study of proinsulin and proglucagon biosynthesis and their metabolic conversion at the subcellular level.