A small-scale method for the isolation of insulin-containing secretory granules from islets of Langerhans

Abstract

A method has been developed which uses small-scale (400 microliter) Percoll gradients and an inexpensive bench-top microcentrifuge for the rapid isolation of insulin-containing secretory granules from islets of Langerhans available from a single rat pancreas. Granule fractions were prepared from homogenates of isolated rat islets by a differential centrifugation step (10 min) to produce a granule-enriched membrane pellet, followed by a further centrifugation (10 min) on a discontinuous Percoll gradient to produce a granule fraction. Measurement of membrane-marker enzyme activities suggested that the yield and purity of granule fractions prepared by this method were comparable to those reported for other methods involving longer centrifugation times in ultracentrifuges. Further purification of the granule fractions by removing lysosomal contamination was achieved by an additional centrifugation (10 min) on another small-scale gradient of higher Percoll concentration. The method proved useful for isolating biosynthetically labeled secretory granule membranes and contents from islets of Langerhans which had been cultured in the presence of 35S-labeled amino acids. The speed and simplicity of this method suggest that it will prove useful in studies requiring the rapid isolation of insulin-containing secretory granules from isolated islets.