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 in gastrointestinal but not skin mast cells, was isolated and its substrate specificity was investigated. Peptide substrates, including angiotensin I, substance P, bradykinin and oxidized insulin B chain were hydrolysed at P1 Phe, Leu or Tyr residues, conforming to the known chymotrypsin-like properties of the enzyme. However, SMCP-1 was found to hydrolyse some chromogenic substrates with P1 Lys and Arg residues. The enzyme also demonstrated trypsin-like activity against protein substrates, cleaving BSA at Lys114-Leu115, Lys238-Val239, Lys260-Tyr261 and Lys376-His377. Bovine fibrinogen beta-chain was cleaved at Lys28-Lys29. To ensure homogeneity of the enzyme, the ratio of chymotrypsin-like to trypsin-like activity was observed; it was found to be constant during purification and between different preparations of SMCP-1. Treatment of SMCP-1 with a range of inhibitors decreased chymotrypsin-like and trypsin-like activities by similar extents, supporting 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 duodenal proteinase, duodenase. It is proposed that SMCP-1 and duodenase represent a new class of ruminant chymases with unusual dual specificities.
Pemberton
A D
AD
Department of Veterinary Clinical Studies, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Scotland, U.K.
Huntley
J F
JF
Miller
H R
HR
D000595:Amino Acid Sequence; D000818:Animals; D002852:Chromatography, Ion Exchange; D053818:Chymases; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism*; D012709:Serum Albumin / Q000378:metabolism; D012756:Sheep; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000494:pharmacology
D004791:Enzyme Inhibitors; D010455:Peptides; D012709:Serum Albumin; D014361:Trypsin Inhibitors; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D053818:Chymases; D014357:Trypsin