Characterization of the cysteine proteinases of the common liver fluke Fasciola hepatica using novel, active-site directed affinity labels.
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.
McGinty
A
A
Division of Biochemistry, School of Biology and Biochemistry, Queen's University of Belfast, Northern Ireland.
Moore
M
M
Halton
D W
DW
Walker
B
B
D000345:Affinity Labels; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives; D003374:Coumarins / Q000378:metabolism; D003470:Culture Media; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D003978:Diazomethane / Q000031:analogs & derivatives; D004151:Dipeptides / Q000378:metabolism; D005210:Fasciola hepatica / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008970:Molecular Weight; D009842:Oligopeptides; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D000345:Affinity Labels; D003374:Coumarins; D003470:Culture Media; D004151:Dipeptides; D005456:Fluorescent Dyes; D009842:Oligopeptides; C064862:biotin-phenylalanyl-alanine diazomethyl ketone; C082374:biotin-phenylalanyl-(S-benzyl)cysteinyl-diazomethane; D003978:Diazomethane; C021159:benzyloxycarbonyl-phenylalanylarginine-4-methylcoumaryl-7-amide; D001710:Biotin; C069012:benzyloxycarbonylarginyl-arginine 4-methylcoumarin-7-ylamide; D003546:Cysteine Endopeptidases