Localization of carboxypeptidase A to the macromolecular heparin proteoglycan-protein complex in secretory granules of rat serosal mast cells.

A macromolecular complex containing carboxypeptidase A, chymase, heparin proteoglycan, and additional proteins resides in the secretory granules of rat mast cells, is released with mast cell activation, and remains concentrated at the extracellular surface of the discharged cells. Partial solubilization and extraction of the secreted complex is achieved by washing the mast cells with 1 M NaCI. The respective net percent release ratios of carboxypeptidase A, heparin, and chymase to β-hexosaminidase from mast cells activated both immunologically and with calcium ionophore are 0.12, 0.14, and 0.12 in physiologic buffer and 0.45, 0.36, and 0.74 after high salt treatment, indicating that carboxypeptidase A, like chymase, is in the secretory granule in ionic linkage with heparin proteoglycan. Analysis of the availability of granule constituents from disrupted mast cells by sucrose density gradient sedimentation performed at different ionic strengths shows that β-hexosaminidase, then chymase, and finally heparin and carboxypeptidase A are sequentially solubilized in 0.15 M NaCI, 1 M NaCI, and 3 M NaCI, respectively. Similarly, during Dowex 1 -x2 chromatography of mast cells disrupted in 1 M NaCI, chymase appears in the 1 M NaCI effluent, whereas heparin and carboxypeptidase A remain bound and co-elute at higher salt concentrations. Thus, the higher salt concentrations needed to solubilize the carboxypeptidase A-heparin complex account for the apparent lower net percent release ratios of these substances, compared with chymase, after a 1 M NaCI wash of activated mast cells. Electrophoresis in sodium dodecyl sulfate (SOS) Polyacrylamide gels of reduced and alkylated denatured mast cell proteins obtained from whole cells and from the calcium ionophore-induced secretions reveals a predominant stained band corresponding to chymase, and four other major bands at m.w. of 30,000, 35,000, 36,000, and 37,000, which are all part of the macromolecular heparin proteoglycan-protein complex in the secretory granule. The close association of the neutral proteases, chymase and carboxypeptidase A, as a macromolecular complex with heparin proteoglycan at the surface of activated mast cells may facilitate their concerted action on proteins at sites of mast cell degranulation.