SHEEP MAST-CELL PROTEINASE-1 - CHARACTERIZATION AS A MEMBER OF A NEW CLASS OF DUAL-SPECIFIC RUMINANT CHYMASES

Sheep mast cell proteinase 1 (SMCP-1), which is abundantly expressed i n gastrointestinal but not skin mast cells, was isolated and its subst rate specificity was investigated. Peptide substrates, including angio tensin I, substance P, bradykinin and oxidized insulin B chain were hy drolysed at P1 Phe, Leu or Tyr residues, conforming to the known chymo trypsin-like properties of the enzyme. However, SMCP-1 was found to hy drolyse some chromogenic substrates with P1 Lys and Arg residues. The enzyme also demonstrated trypsin-like activity against protein substra tes, cleaving BSA at Lys(114)-Leu(115), Lys(238)-Val(239), Lys(260)- T yr(261) and Lys(376)-His(377). Bovine fibrinogen beta-chain was cleave d at Lys(28)-Lys(29). To ensure homogeneity of the enzyme, the ratio o f chymotrypsin-like to trypsin-like activity was observed; it was foun d to be constant during purification and between different preparation s of SMCP-1. Treatment of SMCP-1 with a range of inhibitors decreased chymotrypsin-like and trypsin-like activities by similar extents, supp orting the assertion that both activities are the property of a single enzyme. In terms of activity, and by N-terminal amino acid sequencing , SMCP-1 strongly resembles the similarly dual-specific bovine duodena l proteinase, duodenase. It is proposed that SMCP-1 and duodenase repr esent a new class of ruminant chymases with unusual dual specificities .