The isolation and purification of a dual specific mast cell-derived protease from parasitised caprine jejunal tissue.
A mast cell granule protease has been isolated and purified from nematode-infected caprine jejunal homogenate by FPLC techniques and termed Goat Mast Cell Protease (GMCP). The purification steps were monitored for proteolytic activity against the synthetic substrate carboxybenzoyl-L-lysine thiobenzyl ester (BLT) and the presence of a homogenous protease preparation in the final sample was shown by SDS-PAGE electrophoresis. This protease was compared with enzymatic activity from isolated mucosal mast cells, which demonstrated the putative mast cell-derived source of the purified enzyme. Rabbit antiserum was raised against the protease and through the use of immunohistochemistry and Western blotting techniques the mast cell origin of the protease was confirmed. NH2-Terminal amino acid sequence analysis demonstrated a high degree of homology between GMCP and other previously isolated mast cell proteases including sheep mast cell protease (SMCP). Substrate analysis showed that GMCP also had an unusual dual chymotrypsin-like and trypsin-like activity similar to SMCP and bovine duodenase.
Macaldowie
C N
CN
Moredun Research Institute, Edingburgh.
Mackellar
A
A
Huntley
J F
JF
D000018:Abomasum / Q000201:enzymology / Q000469:parasitology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002417:Cattle; D002846:Chromatography, Affinity; D053818:Chymases; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D015511:Goat Diseases; D006041:Goats / Q000469:parasitology*; D007413:Intestinal Mucosa / Q000201:enzymology* / Q000469:parasitology; D007583:Jejunum / Q000469:parasitology*; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D011817:Rabbits; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D012756:Sheep; D014251:Trichostrongyloidea; D014253:Trichostrongylosis / Q000201:enzymology / Q000662:veterinary*
D000906:Antibodies; D012697:Serine Endopeptidases; D053818:Chymases