Purification of mitochondria and secretory granules isolated from pancreatic beta cells using Percoll and Sephacryl S-1000 superfine

Mitochondria and secretory granules were isolated from beta-cell-rich pancreatic islets of ob/ob mice. Crude fractions obtained by differential centrifugation were subjected to centrifugation on isotonic Percoll. The gradient medium was removed from the resulting fractions by gel filtration on Sephacryl S-1000 Superfine. When compared to the crude fractions, the purified mitochondrial fraction exhibited a 4.5-fold increase in citrate synthase activity, a 70% reduction of lysosomal arylsulfatase, and a 40% decrease of contamination with granular insulin. In the purified secretory granule fraction, the insulin content was as high as 60% of the total protein (albumin standard) with arylsulfatase unchanged and no detectable citrate synthase activity.