Human recombinant mmp 7 proenzyme

The catalytic activity of MMP was analyzed by a peptide cleavage assay using the quenched fluorescent substrate 7-methoxycoumarin-4-yl acetyl-Pro-Leu-Gly-LeuN-3(2,4-dinitrophenyl)-L-2,3 diaminopropionylAla-Arg-NH₂ for MMP-7 and 7-methoxycoumarin-4-yl-Pro-Leu-Ala-L-norvaline-L-2,3 diaminopropionylAla-Arg-NH₂ (a generous gift from Dr. Dong Hae Shin at Ewha Womans University) for MMP-2. Conditioned media (CM) were collected and concentrated using 10 K Amicon^® Ultra Centrifugal Filter Units (Merck Millipore, Burlington, MA, USA). The reactions were performed in a final volume of 200 μL of MMP assay buffer (20 mM Tris HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl₂, 0.5 mM ZnCl₂, 0.001% Brij35) in the presence of 1 μM oligopeptide and 50 ng of human recombinant MMP-7 proenzyme (Sigma-Aldrich, St. Louis, MO, USA) or 50 μL of CM for 1.5 h at 37 °C. Fluorescence was determined at an excitation wavelength of 328 nm and an emission wavelength of 395 nm using a SpectraMax^® i3 plate reader (Molecular Devices, Sunnyvale, CA, USA) [28 (link)].

A monoclonal antibody capable of recognizing human, rat, and mouse SDC2 was produced by AdipoGen Inc. (Incheon, Korea) using the Fc-fused extracellular domain of SDC2 [28 (link)]. A polyclonal antibody that recognizes human, rat, and mouse SDC2 was produced by AbClon (Seoul, Korea) using a human SDC2 extracellular domain peptide. Monoclonal anti-MMP-7 was purchased from R&D Systems (Minneapolis, MN, USA). The quenched fluorescence MMP-7 substrate, (7-methoxycoumarin-4-yl) acetyl-Pro-Leu-Gly-LeuN-3(2,4-dinitrophenyl)-L-2,3 diaminopropionyl Ala-Arg-NH₂, was purchased from Bachem (Bubendorf, Switzerland). Human recombinant MMP-7 proenzyme was purchased from Sigma-Aldrich (St. Louis, MO, USA). Oregon-Green-488-conjugated gelatin from pig skin was purchased from Invitrogen (Waltham, MA, USA).