Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level: I. Fractionation procedure and characterization of the subcellular fractions

Abstract

Anglerfish islets were homogenized in 0.25 M sucrose and separated into 7 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 EM 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 2 of the 7 subcellular fractions contain primarily mitochondria, and that 2 others consist almost exclusively of secretory granules. A 5th fraction contains rough and smooth microsomal vesicles. The remaining 2 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 suitable for study of proinsulin and proglucagon biosynthesis and their metabolic conversion at the subcellular level.