BIOCHEMICAL AND IMMUNOLOGICAL CHARACTERIZATION OF MULTIPLE GLYCOFORMSOF MOUSE MAST-CELL PROTEASE-1 - COMPARISON WITH AN ISOLATED MURINE SEROSAL MAST-CELL PROTEASE (MMCP-4)

Five highly soluble, chymotrypsin-like, neutral serine proteases, with molecular masses in the range 30-33 kDa, were isolated from Trichinel la spiralis-infected mouse small intestine. These enzymes were closely related antigenically on Western blotting and by Ouchterlony double d iffusion using a polyclonal, cross-absorbed, sheep antibody raised aga inst mouse mast cell protease-1 (MMCP-1), and, on the basis of N-termi nal amino acid sequence analysis, were identified as variant forms of MMCP-1. Substrate and inhibitor analysis confirmed that the five varia nts (MMCP-1 A-E) had similar characteristics, although highly signific ant (P = 0.025 to P < 0.0001) variations in K(m) and k(cat.) were dete cted. Against human alpha1-proteinase inhibitor the K(i) for MMCP-1C ( 45 pM) was significantly (P < 0.0001) greater than those for the other proteases (0.76-2.2 pM). The differences in electrophoretic mobility are probably a result of variable glycosylation, since removal of N-li nked carbohydrate produced a polypeptide of approx. 28 kDa in each cas e which was, like the native enzyme, immunoreactive on Western blottin g. A much less soluble 28 kDa enzyme was isolated from serosal mast ce lls and identified as MMCP-4 by N-terminal amino acid sequencing. Like MMCP-1 it has chymotrypsin-like substrate specificities with activity at neutral pH. However, it was anti-genically distinct from MMCP-1 an d, using sheep anti-MMCP-1, was not detected on Western blotting or by Ouchterlony double diffusion, e.l.i.s.a. or immunohistochemistry. Thi s last technique established that the MMCP-1 variants were uniquely pr esent in enteric mast cells, thereby providing a highly selective mean s of distinguishing the mucosal and connective tissue mast cell subset s in the mouse.