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Identification and characterization of the cysteine and serine proteinases of the trematode, Haplometra cylindracea and determination of their haemoglobinase activity

Published online by Cambridge University Press:  06 April 2009

S. J. Hawthorne ,
D. W. Halton  and
B. Walker
S. J. Hawthorne
Affiliation:
Divisions of Biochemistry, The Queen's University of Belfast, Belfast BT7 INN, Northern Ireland, UK
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 INN, Northern Ireland, UK
B. Walker
Affiliation:
Divisions of Biochemistry, The Queen's University of Belfast, Belfast BT7 INN, Northern Ireland, UK
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Summary

The excreted/secreted proteinases of Haplometra cylindracea maintained in vitro, were found to hydrolyse the fluorogenic substrates, Z-ArgArg-NHMec and Z-PheArg-NHMec. This activity was shown to be typically that of cysteine proteinases, as turn-over of both substrates could be blocked by pre-incubation with peptidyl diazomethyl ketones. The biotinylated affinity reagent, biotin-Phe Ala-DMK, used in combination with Z-PheTyr(OBut)-DMK, was employed for the labelling and characterization of these cysteine proteinase activities. Three cathepsin B-like species were detected, with molecular weights of 48, 22–23 and 14 kDa, together with a cathepsin L-like enzyme, with a molecular weight of 55 kDa. The proteinases were also found to have hydrolytic activity towards the substrate, Z-GlyGlyArg-NHMec, which could be blocked by pre-incubation with either of the serine proteinase-selective reagents, Z-ArgP(OPh)2, or biotin-LysP(OPh)2, showing the activity to be trypsin-like. Using the biotinylated affinity label to characterize the trypsin-like enzymes revealed two molecular species with molecular weights of 20 and 24 kDa.

Keywords

Haplometra cylindraceaTrematodaproteinasescysteine proteinasesserine proteinaseshaemoglobinase activity
Type
Research Article
Information
Parasitology , Volume 108 , Issue 5 , June 1994 , pp. 595 - 601
Copyright
Copyright © Cambridge University Press 1994

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