A preliminary analysis of proteolytic activity of excretory-secretory products from Cyathostominea.
The excretory-secretory product (ESP) derived from Cyathostominea in vitro was assessed, in terms of subunit composition, and proteolytic activity using as substrates azocasein and two synthetic fluorogenic peptides. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved 13 subunits, and the presence of the protein cysteine proteinase activator dithiothreitol (DTT) revealed 21 subunits. DTT also enhanced azocaseinolysis, and hydrolysis of carbobenzoxy-phenylalanyl-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-NHMec) and carbobenzoxy-arginyl-arginine-7-amido-4-methylcoumarin (Z-Arg-Arg-NHMec). At the optimum pH of 5.5, hydrolysis of Z-Phe-Arg-NHMec was three-fold greater than that of Z-Arg-Arg-NHMec suggesting that the proteolytic specificities of the ESP are more like those of papain or cathepsin L, rather than cathepsin B. In SDS-PAGE gelatin gels, DTT was a requirement for proteolysis by the ESP. Optimum resolution was at pH=5.5, resolving six bands ranging from 114-20kDa. Cysteine proteinase inhibitors abolished all gelatinolytic activity at the pH values tested. Such data indicate the presence of cysteine-class proteinases in the ESP of Cyathostominea.
Kinsella
Jane L
JL
Department of Zoology, University College Dublin, Belfield, Ireland.
Ralph Lichtenfels
J
J
Ryan
Michael F
MF
D000818:Animals; D002364:Caseins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004789:Enzyme Activation; D005260:Female; D005456:Fluorescent Dyes; D005780:Gelatin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D008297:Male; D012680:Sensitivity and Specificity; D013320:Strongyloidea / Q000201:enzymology*; D013379:Substrate Specificity
D002364:Caseins; D015853:Cysteine Proteinase Inhibitors; D005456:Fluorescent Dyes; C026018:azocasein; D005780:Gelatin; D003546:Cysteine Endopeptidases; D004229:Dithiothreitol