Mouse mast cell protease (mMCP) 6 and mMCP-7 are homologous tryptases stored in granules as macromolecular complexes with heparin and/or chondroitin sulfate E containing serglycin proteoglycans. When promMCP-7 and pseudozymogen forms of this tryptase and mMCP-6 were separately expressed in insect cells, all three recombinant proteins were secreted into the conditioned medium as properly folded, enzymatically inactive 33-kDa monomers. However, when their propeptides were removed, mMCP-6 and mMCP-7 became enzymatically active and spontaneously assumed an ϳ150-kDa tetramer structure. Heparin was not required for this structural change. When incubated at 37°C, recombinant mMCP-7 progressively lost its enzymatic activity in a time-dependent manner. Its N-linked glycans helped regulate the thermal stability of mMCP-7. However, the ability of this tryptase to form the enzymatically active tetramer was more dependent on a highly conserved Trp-rich domain on its surface. Although recombinant mMCP-6 and mMCP-7 preferred to form homotypic tetramers, these tryptases readily formed heterotypic tetramers in vitro. This latter finding indicates that the tetramer structural unit is a novel way the mast cell uses to assemble varied combinations of tryptases. Mouse mast cell (MC)1 protease (mMCP) 6 (1, 2) and mMCP-7 (3, 4) are homologous tryptases stored in abundance in their mature forms in the acidic secretory granules of numerous populations of MCs ionically bound to heparin and/or chondroitin sulfate E containing serglycin proteoglycans (1, 5, 6). Both tryptases are exocytosed when MCs are activated via their high affinity immunoglobulin (Ig) E receptors. mMCP-7 inhibits the formation of fibrin/platelet clots (7), whereas mMCP-6 induces the extravasation of neutrophils (8). Thus, the two tryptases often work in concert in the acute phases of MC-mediated inflammatory responses.A small amount of exocytosed mMCP-7 is retained in tissues for Ͼ1 h (5). However, most exocytosed mMCP-7 dissociates from its exocytosed protease/proteoglycan macromolecular complex in the extracellular matrix to allow the rapid diffusion of this tryptase away from the MC (5, 6). Because its proteoglycan-binding domain has more Lys and Arg residues than the corresponding domain in mMCP-7, exocytosed mMCP-6 cannot dissociate easily from its serglycin proteoglycan at neutral pH. Thus, due to its Ͼ10 million-dalton size, very little of the exocytosed mMCP-6/proteoglycan complex is able to enter the circulation.Pancreatic trypsin and most other serine proteases are enzymatically active in their ϳ30-kDa monomer states. Thus, it was a surprise when Schwartz and co-workers (9, 10) and then others (11-16) discovered that human MC tryptases purified from different tissues exist as tetramers. While it has been proposed that heparin is needed for human lung-and skinderived tryptases to form stable, enzymatically active tetramers (10, 14 -16), mMCP-7 is able to circulate in the blood of the V3 mastocytosis mouse for Ͼ1 h as an enzymatically active, homotypic tetrame...