Demonstration of binding sites for divalent and trivalent ions on the outer surface of chromaffin-granule membranes

1. Trivalent ions Tb3+, Eu3+ and La3+ aggregate chromaffin granules and produce structural changes in the core material. These ions also stain the outer (cytoplasmic) surface of the granule membrane in the presence of PO43+ ions and inhibit OSO4 staining. The electron-dense patches of TbPO4 complex are distributed in a non-random fashion. 2. Tb3+ also functions as a fluorescent membrane probe for divalent ion binding sites on the granule membrane. Using the enhancement of Tb3+ fluorescence upon binding, a Kd for Tb3+ of approximately 15 micronM was measured. Ca2+ and Mg2+ are competitive inhibitors of this binding while Na+ and K+ had no effect. Results suggest that the fluorescent Tb3+ -binding site is a protein. 3. Tb3+ also binds to mitochondria and other contaminants as judged by electron microscopy. However purified mitochondria show qualitatively different binding of Tb3+ by fluorescence. 4. A model for the location of divalent ion binding sites on the granule membrane and the results are discussed in terms of requirements for the participation of these sites in granule exocytosis in vivo.