COMPARISON OF NATIVE MATRIX METALLOPROTEINASES AND THEIR RECOMBINANT CATALYTIC DOMAINS USING A NOVEL RADIOMETRIC ASSAY

A novel radiometric assay was developed for human fibroblast collagena se (matrix metalloproteinase-1, MMP-1), stromelysin (MMP-3), and their recombinant catalytic domains. Using this assay we were able to compa re the native MMPs with the respective catalytic domains in terms of i nhibitor affinities and peptide hydrolysis. The assay works on the sam e principle as an assay developed for carboxypeptidase (Rossier et al. , Anal. Biochem. 1989, 178, 27-31) and is based on a synthetic peptide substrate, -Leu-Ala-Leu-Trp-NH(CH2)(4)N(CH3)(2)(bnzPLALW-NX). The gen eration of product is measured by selective solvent extraction of radi oactive product directly into scintillation cocktail; the entire assay , including the radioactivity measurement, is completed in a single 1- ml tube (96-well format) without removal or transfer of phases. Result s of steady-state measurements demonstrated that peptide hydrolysis fo llows Michaelis-Menten kinetics with the fibroblast MMPs and their C-t erminal deleted forms. The kinetic constants for hydrolysis of bnzPLAL W-NX, and for inhibition by actinonin, a natural peptide-hydroxamate, are essentially the same for the native and the C-terminally deleted M MPs. (C) 1994 Academic Press, Inc.