Significantly more calcium per gram protein was found in a relatively pure granule fraction isolated from fresh bovine adrenal medulla than in predominantly mitochondrial fractions isolated from the same tissue. Sixty-four and 55% of the calcium associated with chromafh granule and mitochondrial fractions, respectively, was released into the supernatant upon lowering the tonicity of the medium. The per cent calcium released by this procedure was significantly greater for; granules than for mitochondria (p < 0.05). The amount of calcium per gram protein released into the supernatant also was greater in granule fractions than in mitochondrial fractions (p < 0.05). These data, coupled with a previous report that 10+ M EDTA does not markedly decrease the calcium content of whole granules, indicate that the excess calcium of the granule fractions relative to the mitochondrial fractions is maintained within the particles of that fraction. The functional significance of the relatively large amount of calcium in chroma* granules is not clear. The presence of 150 mM sodium chloride or potassium chloride decreases calcium binding by granule or mitochondrial fragments incubated in 2.2 mM calcium chloride in 0.2 M Tris, pH 7, by about 50%. EDTA, M, removes all but a small residual of the calcium associated with the granule or mitochondrial fragments whereas lowering the concentration of Tris increases calcium binding to about the same extent in both these subcellular fractions. The calcium-binding properties of granule and mitochondrial fragments therefore appear to be quantitatively and qualitatively similar. Inhibition of catecholamine release by relatively high concentrations of sodium may be explained by competitive inhibition of calcium binding.
Calcium binding by granule fragments decreases with an increase in hydrogen ion concentration.
More calcium per gram protein was reported to be contained in the chromafiln granule fraction of bovine adrenal medulla than in any other subcellular fraction of this tissue (Borowitz et al., '65). This h ding is conhned in this study, and extended to indicate where in the granule the calcium is located.
Although the function of the relatively high concentration of calcium in chromaf-Bn granule fractions is not clear, calcium is known to be essential for catecholamine release (Douglas and Rubin, '63). Since calcium was reported to be taken up by cells of cat adrenal medulla during catecholamine liberation (Douglas and Poisner, '62), it appeared that binding of calcium to catecholamine-containing granules might be of importance in the release process. For this reason, a study of calcium binding, under various conditions, by fragments of granules from bovine adrenal medulla was undertaken. Calcium binding by mitochondrial fragments from the same tissue was measured for comparison.