Comparison of native matrix metalloproteinases and their recombinant catalytic domains using a novel radiometric assay.
A novel radiometric assay was developed for human fibroblast collagenase (matrix metalloproteinase-1, MMP-1), stromelysin (MMP-3), and their recombinant catalytic domains. Using this assay we were able to compare the native MMPs with the respective catalytic domains in terms of inhibitor affinities and peptide hydrolysis. The assay works on the same principle as an assay developed for carboxypeptidase (Rossier et al., Anal. Biochem. 1989, 178, 27-31) and is based on a synthetic peptide substrate, [1-benzoyl-14C]benzoyl-Pro-Leu-Ala-Leu-Trp- NH(CH2)4N(CH3)2(bnzPLALW-NX). The generation of product is measured by selective solvent extraction of radioactive 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. Results of steady-state measurements demonstrated that peptide hydrolysis follows Michaelis-Menten kinetics with the fibroblast MMPs and their C-terminal deleted forms. The kinetic constants for hydrolysis of bnz-PLALW-NX, and for inhibition by actinonin, a natural peptide-hydroxamate, are essentially the same for the native and the C-terminally deleted MMPs.
Brownell
J
J
Sterling Winthrop Pharmaceuticals Research Division, Collegeville, Pennsylvania 19426-0900.
Earley
W
W
Kunec
E
E
Morgan
B A
BA
Olyslager
B
B
Wahl
R C
RC
Houck
D R
DR
D001665:Binding Sites; D002384:Catalysis; D017364:Collagenases / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D006877:Hydroxamic Acids / Q000494:pharmacology; D007700:Kinetics; D020781:Matrix Metalloproteinase 1; D019278:Matrix Metalloproteinase 3; D061965:Matrix Metalloproteinase Inhibitors; D008666:Metalloendopeptidases / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011874:Radiometry; D011994:Recombinant Proteins / Q000378:metabolism
D006877:Hydroxamic Acids; D061965:Matrix Metalloproteinase Inhibitors; D010455:Peptides; D011994:Recombinant Proteins; D017364:Collagenases; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D020781:Matrix Metalloproteinase 1; C010531:actinonin