Salt dependency of chromaffin granule aggregation by annexin II tetramer

Annexin II tetramer (AIIt) is a Ca2+ and phospholipid binding protein that has been shown to reconstitute secretion in permeabilized adrenal medulla cells. In the present study, we have characterized the interactions of AIIt with biological membranes using isolated adrenal medulla secretory granules as a model system. Without added salt, maximal binding of AIIt to chromaffin granules occurred in the absence of AIIt-dependent chromaffin granule aggregation, whereas increasing the osmolality of the reaction mixture with sucrose did not activate AIIt-dependent chromaffin granule aggregation. As the KCl or potassium glutamate concentration of the reaction mixture was increased to between 30 and 50 mM salt, AIIt-dependent chromaffin granule aggregation increased to a maximum, while AIIt binding to chromaffin granules decreased. As the salt concentration was increased from 50 to 150 mM, both AIIt-dependent chromaffin granule aggregation and the binding of AIIt to chromaffin granules were decreased. Furthermore, at optimal salt concentration, KCl and potassium glutamate activated AIIt-dependent aggregation of chromaffin granules to maximum values of about 210% and 195% of control, respectively, whereas potassium phosphate supported AIIt-dependent aggregation of chromaffin granules to only 120% of control. The concentration of AIIt for half-maximal binding to chromaffin granules without added salt or at 50 mM KCl was 0.163 +/- 0.007 (mean +/- SD, n = 3) or 0.173 +/- 0.034 microM AIIt (mean +/- SD, n = 3), respectively, and binding of AIIt to chromaffin granules was not measurable at 150 mM KCl. In contrast, at 50 mM KCl, half-maximal AIIt-dependent chromaffin granule aggregation required 0.171 +/- 0.001 microM AIIt (mean +/- SD, n = 3) and was not measurable without added salt or in the presence of 150 mM KCl. Without added salt, at 50 mM KCl, or at 150 mM KCl, the Ca2+ concentrations for half-maximal aggregation of chromaffin granules and the maximal extent of chromaffin granule aggregation (Amax) were pCa2+ = 3.79 +/- 0.062 (mean +/- SD, n = 3) and Amax = 127% of control, pCa2+ = 6.07 +/- 0.021 (mean +/- SD, n = 3) and Amax = 185% of control, or pCa2+ 4.41 +/- 0.07 (mean +/- SD, n = 3) and Amax = 156% of control, respectively. The stimulation of chromaffin granule aggregation activity and the chromaffin granule binding activity of AIIt was reversible by removal of Ca2+. These results suggest that both ionic strength and salt composition modulate both AIIt-dependent chromaffin granule aggregation and binding to the membranes of these secretory granules.