Active site-dependent initiation at 1/sup 0/C by Chymase (CHY) of rat serosal mast cell (RSMC) exocytosis

Exposure of RSMC (> 95% purity) to isolated CHY (0.5-1.5 U/ml), the major secretory granule protease, at 37/sup 0/C results in exocytosis, determined by the release of a soluble secretory granule enzyme, ..beta..-hexosaminidase. CHY-mediated RSMC exocytosis does not occur at 1/sup 0/C. Exposure of RSMC to CHY or ..cap alpha..-chymotrypsin (CT) at 1/sup 0/C, removal of buffer and resuspension of RSMC in buffer alone at 37/sup 0/C, yields the same exocytosis as direct exposure of RSMC to those chymotryptic enzymes at 37/sup 0/C. Differences in the interaction of CHY and CT with RSMC at 1/sup 0/C and 37/sup 0/C are dose-dependent, not qualitative. Binding to (< 0.5% of input) and dissociation of /sup 125/I-labeled CT (3-8 x 10/sup 8/ cpm/mg) from RSMC, as determined by spinning through oil, was time independent and saturation of specific binding was not achieved, indicating that the observed binding is nonspecific. Diisopropyl fluorophosphate (DFP) and lima bean trypsin inhibitor (LBTI) prevent subsequent exocytosis at 37/sup 0/C only if added within the first 10 min of the interaction of RSMC and CHY at 1/sup 0/C. Maximal CHY-mediated RSMC activation at 1/sup 0/C is achieved within 10 min and addition of DFP and LBTI after this periodmore » does not affect subsequent exocytosis. The dose- and time-dependent inhibition by DFP and LBTI at 1/sup 0/C of CHY initiation of RSMC exocytosis suggest that an enzymatic action of CHY on RSMC at 1/sup 0/C, not a binding reaction, commits RSMC to exocytosis at 37/sup 0/C.« less