Document Detail


A quantitative assay for aggrecanase activity.
MedLine Citation:
PMID:  16522869     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Aggrecanase activities of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteinases were measured with a recombinant aggrecan fragment and two monoclonal antibodies. Recombinant human aggrecan interglobular domain was first incubated in the presence of ADAMTS enzymes. The aggrecan peptide with the N-terminal sequence ARGSVIL released upon hydrolysis was then quantified in an enzyme-linked immunosorbent assay (ELISA) with an anti-neoepitope antibody specific for the N-terminal ARGSVIL sequence and a second anti-aggrecan peptide antibody. For higher sensitivity of the assay, P1-P5 residues of the aggrecanase site within the aggrecan substrate were changed by in vitro mutagenesis. Specific activities of recombinant truncated ADAMTS1 and ADAMTS4 estimated with authentic aggrecan interglobular domain amounted to 2.4 +/- 0.4 and 21.7 +/- 9.5 nmoles hydrolyzed substrate/min.mg, respectively. The values were 10.3 +/- 5.1 and 151.5 +/- 93.5 nmoles/min.mg for hydrolysis of the modified substrate. The aggrecanase activity assay can be used for (1) kinetic characterization of aggrecanase activities of human and animal ADAMTS, (2) screening of inhibitors for aggrecan hydrolyzing ADAMTS, and (3) estimation of aggrecanase activities in biological samples.
Authors:
Horst Will; Matthias Dettloff; Peter Bendzkô; Peter Sveshnikov
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of biomolecular techniques : JBT     Volume:  16     ISSN:  1524-0215     ISO Abbreviation:  J Biomol Tech     Publication Date:  2005 Dec 
Date Detail:
Created Date:  2006-03-08     Completed Date:  2006-04-12     Revised Date:  2013-06-07    
Medline Journal Info:
Nlm Unique ID:  100888641     Medline TA:  J Biomol Tech     Country:  United States    
Other Details:
Languages:  eng     Pagination:  459-72     Citation Subset:  IM    
Affiliation:
Invitek GmbH, Berlin, Germany. proteinexpression@invitek.de
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MeSH Terms
Descriptor/Qualifier:
ADAM Proteins / chemistry,  isolation & purification,  metabolism*
Amino Acid Sequence
Biological Assay / methods
Blotting, Western
Chromatography, Gel
DNA, Complementary / genetics
Endopeptidases / analysis*,  metabolism*
Enzyme-Linked Immunosorbent Assay
Epitopes
Humans
Hydrolysis
Kinetics
Nucleic Acid Amplification Techniques
Polymerase Chain Reaction
Protein Engineering
Protein Structure, Tertiary
Synovial Fluid / enzymology
Chemical
Reg. No./Substance:
0/DNA, Complementary; 0/Epitopes; EC 3.4.-/Endopeptidases; EC 3.4.24.-/ADAM Proteins; EC 3.4.24.-/ADAMTS1 protein, human; EC 3.4.99.-/aggrecanase
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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