Separation of ovine chromaffin granules from lysosomes by successive isoosmolar and hyperosmolar density gradient centrifugation.

Preparations containing ovine chromaffin granules and lysosomes were obtained by differential centrifugation and applied to density gradients. In isoosmolar linear Metrizamide gradients the granules had a lower density than the major portion of the lysosomes (1.05 compared to 1.15); however, in hyperosmolar linear Metrizamide gradients the granule density increased and they migrated close to the lysosomes. The granules separated into two bands on a discontinuous isoosmolar Metrizamide gradient; however, these two bands were similar in terms of granule and lysosomal markers. On a discontinuous hyperosmolar sucrose gradient the granules were more dense than the lysosomes, the reverse of the situation on the Metrizamide gradient. Separation on a discontinuous isoosmolar Metrizamide gradient followed by a 1.8 M sucrose cushion provided a 54-fold purification of granules from lysosomes and similar separations from other subcellular markers. This procedure also provided a 37-fold purification of bovine granules from lysosomes. It was demonstrated that thimet oligopeptidase (EC 3.4.24.15) occurred in the adrenal medulla but is not principally located in the chromaffin granule.