Identification and purification of an adrenal medullary protein (synexin) that causes calcium-dependent aggregation of isolated chromaffin granules

We have isolated from the adrenal medulla a protein which causes the aggregation of isolated chromaffin granules when incubated in the presence of free calcium at concentrations greater than 6 μM. The isolation procedure included precipitation in ammonium sulfate, gel filtration, and hydroxylapatite chromatography. Aggregating activity was assayed using turbidity measurements on granule suspensions. The protein was found to be soluble, heat-labile, and trypsin-sensitive and had an apparent molecular weight of 47,000 when subjected to electrophoresis in gels containing sodium dodecyl sulfate. It did not cause granule aggregation in the presence of magnesium, barium, or strontium, but was activated in a positively cooperative manner by calcium with a 'Hill coefficient' of approximately 2. The aggregation was strongly temperature-dependent below 25°, was stimulated by salts of monovalent ions up to 30 mM, and could be prevented by prior fixation of granules with glutaraldehyde. The protein apparently bound to granules in a calcium-dependent fashion as its activity could be removed from solution by centrifugation of aggregating granules. Electron micrographs of granule aggregates revealed intact granules slightly distorted by planar regions of contact with one another. Viewed in cross-section, the regions of contact between granule membranes had a pentalaminar structure similar to that initially seen between a vesicle and plasma membrane in the process of exocytosis. We have named this protein 'synexin', from the Greek synexis, which means 'meeting'. We suggest that synexin may be the intracellular receptor for calcium in the process of exocytosis from the adrenal medulla, acting to promote close association of granules both with other granules as well as with plasma membranes prior to secretion.