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. Iri the present study, we have characterized the inter actions of AIIt with biological membranes using isolated adrenal medul la secretory granules as a model system. Without added salt, maximal b inding of AIIt to chromaffin granules occurred in the absence of AIIt- dependent chromaffin granule aggregation, whereas increasing the osmol ality of the reaction mixture with sucrose did not activate AIIt-depen dent chromaffin granule aggregation. As the KCI or potassium glutamate concentration of the reaction mixture was increased fo between 30 and 50 mM salt, AIIt-dependent chromaffin granule aggregation increased t o a maximum, while AIIt binding to chromaffin granules decreased. As t he salt concentration was increased from 50 to 150 mM, both AIIt-depen dent chromaffin granule aggregation and the binding of AIIt to chromaf fin granules were decreased. Furthermore, at optimal salt concentratio n, KCl and potassium glutamate activated AIIt-dependent aggregation of chromaffin granules to maximum values of about 210% and 195% of contr ol, respectively, whereas potassium phosphate supported AIIt-dependent aggregation of chromaffin granules to only 120% of control. The conce ntration of AIIt fdr half-maximal binding to chromaffin granules witho ut added salt or at 50 mM KCl was 0.163 +/- 0.007 (mean +/- SD, n = 3) or 0.173 +/- 0.034 mu M AIIt (mean +/- SD, n = 3), respectively, and binding of AIIt to chromaffin granules was not measurable at 150 mM KC l. in contrast, sit 50 mM KCl, half-maximal AIIt-dependent chromaffin granule aggregation required 0.171 +/- 0.001 mu M AIIt (mean +/- SD, n = 3) and was not measurable without added salt oi. in the presence of 150 mM KCI. Without added salt, at 50 mM KCI; or at 150 mM KCl, the C a2+ concentrations for half-maximal aggregation of chromaffin granules and the maximal extent of chromaffin granule aggregation (A(max)) wer e pCa(2+) = 3.79 +/- 0.062 (mean +/- SD, n = 3) and A(max) = 127% of c ontrol, pCa(2+) = 6.07 +/- 0.021 (mean +/- SD, n = 3) and A(max) = 185 % of control, br pCa(2+) 4.41 +/- 0.07 (mean +/- SD, n = 3) and A(max) = 156% of control, respectively. The stimulation of chromaffin granul e aggregation activity and the chromaffin granule binding activity of AIIt was reversible by removal of Ca2+. These results suggest that bot h ionic strength and salt composition modulate both AIIt-dependent chr omaffin granule aggregation and binding to the membranes of these secr etory granules.