A fluorescent peptide substrate for the surface metalloprotease of Leishmania.

Details

Serval ID
serval:BIB_B8E3E6E367ED
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
A fluorescent peptide substrate for the surface metalloprotease of Leishmania.
Journal
Experimental Parasitology
Author(s)
Bouvier J., Schneider P., Malcolm B.
ISSN
0014-4894 (Print)
ISSN-L
0014-4894
Publication state
Published
Issued date
1993
Volume
76
Number
2
Pages
146-155
Language
english
Abstract
A fluorescent oligopeptide substrate for the promastigote surface protease (PSP) of Leishmania was designed using the data reported for the substrate specificity of the enzyme (Bouvier, J., Schneider, P., Etges, R. J., and Bordier, C. 1990. Biochemistry 29, 10113-10119). The indole fluorescence of the tryptophan residue was efficiently quenched through resonance energy transfer by an N-terminal dansyl group located five amino acid residues away. The heptapeptide, dansyl-A-Y-L-K-K-W-V-NH2, was cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 8.8 x 10(6) M-1sec-1. Hydrolysis by the enzyme results in a time-dependent increase of fluorescence intensity of 3.7-fold. Assays can be designed based on the tryptophan fluorescence at 360 nm or by individual product analyses using thin-layer chromatography. The synthetic substrate is readily cleaved by the metalloprotease at the surface of fixed promastigotes. The specificity and sensitivity of such internally quenched fluorescent peptide substrate will facilitate the identification of novel inhibitors for the enzyme and aid in detailed studies on its enzymology.
Keywords
Amino Acid Sequence, Animals, Fluorescent Dyes, Hydrolysis, Kinetics, Leishmania/enzymology, Metalloendopeptidases/metabolism, Molecular Sequence Data, Oligopeptides/chemistry, Oligopeptides/metabolism, Spectrometry, Fluorescence, Substrate Specificity
Pubmed
Web of science
Create date
19/01/2008 17:30
Last modification date
20/08/2019 15:26
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