Document Detail


Fluorescent, internally quenched, peptides for exploring the pH-dependent substrate specificity of cathepsin B.
MedLine Citation:
PMID:  16485313     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Cathepsin B is a cysteine protease that in tumor tissues is localized in both acidic lysosomes and extracellular spaces. It can catalyze the cleavage of peptide bonds by two mechanisms: endoproteolytic attack with a pH optimum around 7.4, and attack from the C-terminus with a pH optimum at 4.5-5.5. In this work, seven fluorescent, internally quenched, decapeptides have been synthesized using the prototypical cathepsin B selective substrate Z-Phe-Arg-AMC as a lead, and used to identify the structural factors determining the susceptibility of peptides to hydrolysis at acidic and neutral pH values. Each peptide differs from the others in one amino acid (residue 6) and contains a highly fluorescent Nma group linked to the alpha-amino function of the N-terminal Orn residue and a Dnp group linked to the side chain of the Lys(8) residue acting as a quencher. Proteolytic cleavage was monitored by measuring the increase of fluorescence at 440 nm upon excitation at 340 nm, and the cleavage sites were determined by HPLC followed by ESI-MS analysis. Peptides containing Ala or Phe at position 6 are good substrates for the enzyme at both pH 5.0 and 7.4. By contrast, those containing Glu, Asp, Lys or Val are not cleaved at all by cathepsin B at pH 7.4, and are poorly hydrolyzed at pH 5.0. These findings provide new information for the rational design of cathepsin B-activated peptide-containing anticancer drugs.
Authors:
Paolo Ruzza; Luigi Quintieri; Alessio Osler; Andrea Calderan; Barbara Biondi; Maura Floreani; Andrea Guiotto; Gianfranco Borin
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Publication Detail:
Type:  In Vitro; Journal Article    
Journal Detail:
Title:  Journal of peptide science : an official publication of the European Peptide Society     Volume:  12     ISSN:  1075-2617     ISO Abbreviation:  J. Pept. Sci.     Publication Date:  2006 Jul 
Date Detail:
Created Date:  2006-06-28     Completed Date:  2006-09-08     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  9506309     Medline TA:  J Pept Sci     Country:  England    
Other Details:
Languages:  eng     Pagination:  455-61     Citation Subset:  IM    
Affiliation:
Institute of Biomolecular Chemistry of CNR, Padua Unit, 35131 Padua, Italy. paolo.ruzza@unipd.it
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MeSH Terms
Descriptor/Qualifier:
Animals
Cathepsin B / chemistry*,  metabolism*
Cattle
Fluorescent Dyes
Hydrogen-Ion Concentration
Kinetics
Oligopeptides / chemical synthesis,  chemistry*,  metabolism*
Protein Conformation
Spectrometry, Fluorescence
Substrate Specificity
Chemical
Reg. No./Substance:
0/Fluorescent Dyes; 0/Oligopeptides; EC 3.4.22.1/Cathepsin B

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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