Specificity of synexin-induced chromaffin granule aggregation

Purification and Mode of Action of Synexin: A Protein Enhancing Calcium‐Induced Membrane Aggregation

“…This is especially important since it has recently been demonstrated that under certain conditions, semipurified synexin also enhanced the subsequent Ca2+-promoted fusion of phospholipid vesicles (Hong et al, 1981) and chromaffin granules (Ed- wards et al, 1974;Moms, 1974;Creutz, 1981). In the present work it was shown that the purified protein significantly enhanced the Ca2+-induced aggregation of phosphatidylserine (PS) and liposomes containing up to 40% phosphatid ylcholine (PC), and, in answer to the criticisms of Dabrow et al (1980), that this could not be explained by extinction changes owing to self-association of synexin molecules in the presence of Ca2+.…”

“…Our results show that there is a marked parallelism between synexin activity, measured either by the enhancement of Ca2+-induced aggregation of CGMs or PS-liposomes, and the enrichment of the 47,000 M, protein through several stages of purification. Except for the original observations of Creutz et al (1978) that hydroxyl apatite-purified synexin will aggregate granules, all other studies on the properties of this protein (Creutz et al, 1979;Morris and Hughes, 1979;Dabrow et al, 1980;Hong et al, 1981; have used Ultragel AcA 34-purified material. Since our calcium precipitate-purified fraction F,p still contained the 62,000 M, contaminant (Fig.…”

“…Following the report of Dabrow et al (1980) that Ca2+-induced aggregation of trypsin-or pronasetreated CGMs was not enhanced by synexin, several attempts were made to repeat these findings. When the membranes were thoroughly washed to remove residual amounts of trypsin, synexin enhancement of Ca2+-induced aggregation was found to be unaffected.…”

“…However, synexin itself, in amounts similar to those used in the assay system, was destroyed by incubation with trypsin (100 pg*ml-' for 1 h at 20°C). It seems likely that the results of Dabrow et al (1980) were due to failure to remove trypsin completely before the addition of synexin. Evidence that trypsin was active in the digestion of the protein in our membranes was provided by the observation that CGMs incubated with 200 p g of trypsin-ml-' tended to flocculate and gave a base-line extinction 50% higher than that of untreated controls.…”