Physicochemical characteristics of insulin secretion granules

Abstract

β-Granules were prepared from micro-dissected pancreatic islets of obese–hyperglycaemic mice. This fraction contained 60% of the insulin, 30% of the cytochrome oxidase, 16% of the acid phosphatase activity and 20% of the protein present in whole islets. The isolated granules retained a heavy metal during fractionation. Optimum conditions for granule stability were low ionic strength and pH6, the granules being unexpectedly fragile at pH7·4. The stability of the granules was unaffected by sucrose in the concentration range 50–320mm, but 1% (w/v) sodium deoxycholate released all insulin. A solubilizing effect was also noted with ATP and citrate. Spinning through 1·6m-sucrose yielded a further purification in relation to mitochondria and acid-phosphatase-carrying particles but virtually no purification in relation to protein. Electron microscopy revealed that the major contaminants were rough-surfaced vesicles and membranes. A separation of granules from acid phosphatase was achieved by phase distribution in polyethylene glycol and dextran. The location of the enzyme to the interphase was so pronounced in systems buffered with lithium phosphate that the technique may be used for future purification of acid-phosphatase-carrying particles from the β-cells.