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Characterization of the cysteine proteinases of the common liver fluke Fasciola hepatica using novel, active-site directed affinity labels

Published online by Cambridge University Press:  06 April 2009

A. McGinty ,
M. Moore ,
D. W. Halton  and
B. Walker
A. McGinty
Affiliation:
Divisions of Biochemistry and Experimental Biology, School of Biology and Biochemistry, Medical Biology Centre, The Queen's University of Belfast, Belfast BT7 1NN, Northern Ireland
M. Moore
Affiliation:
Divisions of Biochemistry and Experimental Biology, School of Biology and Biochemistry, Medical Biology Centre, The Queen's University of Belfast, Belfast BT7 1NN, Northern Ireland
D. W. Halton
Affiliation:
Divisions of Cell and Experimental Biology, School of Biology and Biochemistry, Medical Biology Centre, The Queen's University of Belfast, Belfast BT7 1NN, Northern Ireland
B. Walker
Affiliation:
Divisions of Biochemistry and Experimental Biology, School of Biology and Biochemistry, Medical Biology Centre, The Queen's University of Belfast, Belfast BT7 1NN, Northern Ireland
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Summary

The excreted/secreted proteinases of adult and juvenile Fasciola hepatica maintained in vitro were found to hydrolyse the fluorogenic substrates Cbz-Phe-Arg- and Cbz-Arg-Arg-NHMec. This activity was demonstrated to have a classical cysteine proteinase inhibitor profile, with turn-over of both substrates being blocked by pre-incubation with E64 and peptidyl diazomethanes. The Cbz-Arg-Arg-NHMec hydrolysing activity of the mature fluke exhibited an alkaline stability not characteristic of its mammalian lysosomal counterparts. Further, the biotinylated affinity reagents biotin-Phe-Ala- CHN2 and biotin-Phe-Cys(SBzyl)-CHN2 were used to label and characterize these cysteine proteinases in terms of apparent molecular weight and subsite specificity. Adult fluke media were found to contain four species of molecular weights 66, 58, 50 and 25–26 kDa; juvenile media contained three species of molecular weights 66, 54 and 25–26 kDa. The major 25–26 kDa cysteine proteinase common to both stages was shown to have a subsite specificity similar to that of mammalian cathepsin B.

Keywords

Fasciola hepaticacysteine proteinasebiotinylated affinity labels
Type
Research Article
Information
Parasitology , Volume 106 , Issue 5 , June 1993 , pp. 487 - 493
Copyright
Copyright © Cambridge University Press 1993

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