Active Tissue Inhibitors Of Metalloproteinase 3 (TIMP3)

Tissue Inhibitors Of Metalloproteinase 3 (TIMP3) is an protein belongs to the tissue inhibitor of metalloproteinases family. They are inhibitors of the matrix metalloproteinases. TIMP-3 is the only member of the TIMP family which is found exclusively in the extracellular matrix (ECM). It is regulated in a cell cycle-dependent fashion in certain cell types and may serve as a marker for terminal differentiation. The activity of recombinant human TIMP3 was measured by its ability to inhibit recombinant human MMP2 cleavage of a fluorogenic peptide substrate Mca-PLGL-Dpa-AR-NH2 in the assay buffer 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35 (v/v), pH 7.5. The rhMMP-2 (APA100Hu61) was activated with 1 mM APMA at 37 °C for 1h. Then 16 μl 126 ug/mL rhTIMP-3, 25.6 µL of 100 ug/ml rhMMP-2 and 118.4 µL of assay buffer was incubated for 2 hours at 37 °C, including a control (in duplicate) containing assay buffer and the diluted rhMMP-2. The mixtures was diluted 5 fold in assay buffer followed by adding 50 ul 20 uM substrate, including a control containing assay buffer and substrate. Then read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes. Under these conditions, the enzyme amount of 50% inhibition of rhMMP-2 activity per minute is defined as a unit. The specific activity of TIMP3 is >6000 U/mg.

Tissue Inhibitors Of Metalloproteinase 3 (TIMP3) is an protein belongs to the tissue inhibitor of metalloproteinases family. They are inhibitors of the matrix metalloproteinases. TIMP-3 is the only member of the TIMP family which is found exclusively in the extracellular matrix (ECM). It is regulated in a cell cycle-dependent fashion in certain cell types and may serve as a marker for terminal differentiation. The activity of recombinant human TIMP3 was measured by its ability to inhibit rhMMP2 cleavage of a fluorogenic peptide substrate MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 in the assay buffer 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5. rhMMP2 was diluted to 100 ug/ml and activated with 1 mM APMA at 37 °C for 1 hour and rhTIMP3 (MW: 25.28 KD) was diluted to different concentrations with the assay buffer. Mix 8 µl of rhTIMP3 curve dilutions, 12.8 µl of activated rhMMP-2, and 59.2 µl of assay buffer, including a control containing assay buffer and the diluted rhMMP-2 and incubate the reactions for 2 hours at 37 °C. Loading 50 µl of the incubated mixtures which were diluted five-fold in assay buffer into empty wells of a plate, and start the reaction by adding 50 µl of 20 µM substrate. Include a substrate blank containing 50 µl of assay buffer and 50 µl of 20 µM substrate. Then read at excitiation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes. The result was shown in Figure 1 and it was obvious that recombinant human TIMP3 significantly decreased rhMMP2 activity. The inhibition IC50 was <15 nM.