ISOLATION AND PROPERTIES OF SECRETORY GRANULES FROM RAT ISLETS OF LANGERHANS II. Ultrastructure of the Beta Granule

Beta granules isolated from rat islets of Langerhans and subjected only to phosphotungstic acid had, in negatively stained images, a 50-A periodicity . This periodicity was also observed in thin-section profiles of beta granules in intact cells . In shadowed preparations, the granules were spherical in shape and had irregular edges and surface structure . The presence of such a periodicity in the beta granule indicates that its matrix may be composed of a crystalline material . The isolation of a fraction of secretory granules from isolated islets of the rat pancreas (Howell, Fink, and Lacy, 1969) has made possible further characterization of the morphological properties of the isolated granules by methods not applicable to granules retained within the intact cell . MATERIALS AND METHODS In most experiments, the washed pellet of the secretory granule fraction, prepared by density gradient centrifugation (Howell et al ., 1969), was resuspended in 0.2 ml of a cold medium containing 160 mM KCI, 5 mm NaCl, 0 .5 MM MgC1 2 , and 5 mm sodium phosphate, pH 6 .0, and maintained at ice-bath temperature while samples were withdrawn . Grids were prepared as soon as the pellet had been resuspended, since the insulin content of the granules becomes progressively solubilized with time after suspension (Howell, Young, and Lacy, 1969) . All grids were prepared within 10 min of resuspension . In a single experiment, the pellet was resuspended in 2 .5% glutaraldehyde in 0 .15 M phosphate buffer, pH 7 .5 . In a further experiment, the pellet was resuspended in 0 .3 M sucrose, buffered at pH 6 .0 with 0.005 M phosphate buffer . For negatively stained preparations, a small drop of suspension was placed on a collodion-carboncoated 400-mesh copper grid, allowed to sit for 1 min, then drained by contact with filter paper . A drop of 2% % phosphotungstic acid, pH 6 .0, was placed on the grid and, after a minute, was drained in the same way . The first grid thus prepared was immediately placed in the vacuum chamber of the electron microscope ; other grids were prepared and allowed to dry in a Petri dish containing a desiccant . For shadow-casted preparations, after a drop of the suspension had been drained from the grid, the grid was flushed several times with distilled water for removal of salts . Some of the grids, while still wet, were subjected to osmium vapors for several minutes ; others were allowed to dry in a Petri dish containing a desiccant . The grids were shadowed with platinum at an angle of 10°, or alternatively shadowed with platinum during continuous rotation of the grid (rotational shadowing) . Secretory granule pellets, isolated islets, and pancreatic tissue were fixed in 2 .5%/%, glutaraldehyde followed by osmium tetroxide solution, dehydrated in ethyl alcohol, and embedded in an epoxy resin according to Lockwood's procedure (Lockwood, 1964) . The thin sections were stained with uranyl on F ebuary 1, 2013 jcb.rress.org D ow nladed fom Published April 1, 1969