Characterisation of proteolytic activity of excretory-secretory products from adult Strongylus vulgaris.
An excretory-secretory (ES) preparation derived from adult Strongylus vulgaris in vitro was assessed for proteolytic activity using azocasein and synthetic, fluorogenic, peptide substrates. Fractionation was by molecular sieve fast protein liquid chromatography (molecular sieve FPLC) and resolution by gelatin-substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis (gelatin-substrate SDS-PAGE). The cysteine proteinase activator, dithiothreitol (DTT), enhanced azocaseinolysis and hydrolysis of carbobenzoxy-phenylalanyl-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-NMec) by the ES preparation and was a requirement for the detection of carbobenzoxy-arginyl-arginine-7-amido-4-methylcoumarin (Z-Arg-Arg-NMec) hydrolysis. Assays of FPLC-eluted fractions, with DTT, detected a broad peak of azocaseinolytic activity (22-24 kDa) and two peaks (24 and 18 kDa) of hydrolysis using the synthetic substrates. Hydrolysis by these peaks of Z-Phe-Arg-NMec was 50-fold greater than that of Z-Arg-Arg-NMec suggesting that their specificities are more like papain or cathepsin L rather than cathepsin B. In gelatin-substrate SDS-PAGE, DTT was required to detect proteolysis by the ES preparation which was optimal at pH 6.0 and resolved into eight bands (87-29 kDa). Cysteine proteinase inhibitors were the most effective in all assays. Collectively, these data indicate that cysteine-class proteolytic activity predominates in the ES preparation of adult S. vulgaris.
Caffrey
C R
CR
Department of Zoology, University College Dublin, Belfield, Ireland.
Ryan
M F
MF
D000818:Animals; D002364:Caseins / Q000378:metabolism; D002850:Chromatography, Gel / Q000662:veterinary; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D006736:Horses; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D013319:Strongyle Infections, Equine / Q000469:parasitology; D013323:Strongylus / Q000201:enzymology*
D002364:Caseins; D005456:Fluorescent Dyes; D010455:Peptides; C026018:azocasein; D003546:Cysteine Endopeptidases; D004229:Dithiothreitol