A Preliminary Analysis of Proteolytic Activity of Excretory-Secretory Products from Cyathostominea

Authors

Jane L. Kinsella, University College Dublin
J. Ralph Lichtenfels, Animal Parasitic Disease Lab, ARS, United States Department of AgricultureFollow
Michael F. Ryan, University College DublinFollow

Date of this Version

2002

Comments

Published in Veterinary Parasitology (2002) 107: 73-83.

Abstract

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.DTTalso 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 114–20 kDa. 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.