A microplate assay specific for the enzyme aggrecanase

Anal Biochem. 2003 Mar 15;314(2):260-5. doi: 10.1016/s0003-2697(02)00638-3.

Abstract

We have identified a 41-residue peptide, bracketing the aggrecanase cleavage site of aggrecan, that serves as a specific substrate for this enzyme family. Biotinylation of the peptide allowed its immobilization onto streptavidin-coated plates. Aggrecanase-mediated hydrolysis resulted in an immobilized product that reveals an N-terminal neoepitope, recognized by the specific antibody BC-3. This assay is highly specific for aggrecanases; MMPs were inactive in this assay. Reduction of the peptide size below 30 amino acids resulted in a significant diminution of activity. Using the immobilized 41-residue peptide as a substrate, we have developed a 96-well microplate-based assay that can be conveniently used for high-throughput screening of samples for aggrecanase activity and for discovery of inhibitors of aggrecanase activity.

Publication types

  • Comparative Study

MeSH terms

  • Aggrecans
  • Amino Acid Sequence
  • Animals
  • Binding Sites / genetics
  • Biotinylation
  • Cartilage / enzymology
  • Cattle
  • Chromatography, High Pressure Liquid
  • Culture Media, Conditioned / pharmacology
  • Edetic Acid / pharmacology
  • Endopeptidases / genetics
  • Endopeptidases / metabolism*
  • Enzyme Activation / drug effects
  • Enzyme Inhibitors / pharmacology
  • Extracellular Matrix Proteins*
  • Hydrolysis
  • Lectins, C-Type
  • Matrix Metalloproteinases / metabolism
  • Molecular Sequence Data
  • Phenanthrolines / pharmacology
  • Protease Inhibitors / pharmacology
  • Proteoglycans / metabolism*
  • Substrate Specificity
  • Time Factors

Substances

  • Aggrecans
  • Culture Media, Conditioned
  • Enzyme Inhibitors
  • Extracellular Matrix Proteins
  • Lectins, C-Type
  • Phenanthrolines
  • Protease Inhibitors
  • Proteoglycans
  • Edetic Acid
  • Endopeptidases
  • Matrix Metalloproteinases
  • aggrecanase
  • 1,10-phenanthroline