ISOLATION OF AN INSULIN SECRETION GRANULE FRACTION

Abstract

Goosefish islets were homogenized in 0.25 M sucrose and separated into nuclear, mitochondrial + secretion granule, microsomal, and supernatant fractions. Eighty per cent of the cytochrome oxidase activity and 75 per cent of the bioassayed insulin activity were found in the mitochondrial + secretion granule fraction (6000 g for 10 minutes). The mitochondrial + secretion granule fraction was further subfractionated by centrifugation (2 hours at 100,000 g and 0 degrees C) using a continuous linear density gradient 1.0-2.0 M sucrose). Eighteen to 20 subfractions were collected by piercing the bottom of the tube and collecting drops. The total protein was distributed into a bimodal curve consisting of a high density component, which contained 90 per cent of the insulin (secretion granules), and a lower density component, which contained the cytochrome oxidase activity (mitochondria).