Jr. Doughty et al., A STROMELYSIN ASSAY FOR THE ASSESSMENT OF METALLOPROTEASE INHIBITORS ON HUMAN AGGREGATED PROTEOGLYCAN, Agents and actions, 39, 1993, pp. 30000151-30000153
Human proteoglycan was aggregated to an immobilized hyaluronan solid p
hase on a 96-well ELISA plate. This complex was then degraded by recom
binant human stromelysin. The remaining proteoglycan fragments were de
tected using a monoclonal antibody probe directed against the chondroi
tin sulfate (CS) region of the core protein. Stromelysin degraded the
aggregate in a time and dose dependent manner as reflected by the loss
of the CS epitope. Assay sensitivity was 0.125 U/well with total loss
of the CS epitope occurring at 4 U/well. o-phenanthroline (IC50 = 52
muM) and U24522 (IC50 = 9 muM) inhibited degradation, while phosphoram
idon did not. Serine and cysteine protease inhibitors had no effect. A
comparative analysis of this assay with a reference method, substance
P assay, gave similar inhibitor profiles. The use of aggregated human
proteoglycan (native conformation) as a substrate, may better reflect
how stromelysin inhibitors behave in the presence of complex substrat
es such as cartilage matrix.