• Purity

  >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie? Blue Staining.

• Endotoxin Level

  <0.10 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave a fluorogenic peptide substrate Mca-KPLGL-Dpa-AR-NH    ₂ (Catalog # ). The specific activity is >100 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Mouse myeloma cell line, NS0-derived Ala18-Cys462

• Accession #

• N-terminal Sequence    
  Analysis

  Ala18

• Structure / Form

  Pro form

• Predicted Molecular Mass

  52 kDa

• SDS-PAGE

  50-62 kDa, reducing conditions

Assay Procedure

Materials

• Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5
  (TCNB) + 5 μM ZnCl₂

• Assay  Buffer: 50 mM Tris, 10 mM CaCl₂,150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)

• Recombinant  Mouse MMP-12 (rmMMP-12) (Catalog # 3467-MPB)

• p-aminophenylmercuric acetate (APMA) (Sigma, Catalog # A9563), 100 mM stock in DMSO

• Substrate: MCA-Lys-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2 (Catalog # ), 2 mM stock in DMSO

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent

1. Dilute rmMMP-12 to 100 μg/mL with Activation Buffer containing 1 mM APMA.

2. To activate, incubate rmMMP-12 at 37 °C for 24 hours.

3. Dilute activated rmMMP-12 to 2 μg/mL in Assay Buffer.

4. Dilute Substrate to 20 μM in Assay Buffer.

5. Load 50 μL of 2 μg/mL rmMMP-12 to wells, and start the reaction by adding 50 μL of 20 μM Substrate. Include a Substrate blank containing 50 μL Assay Buffer and 50 μL of 20 μM.

6. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.

7. Calculate specific activity:
    
   

     *Adjusted for Substrate Blank.                                                                                      

**Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

Per Well:

• rmMMP-12: 0.1 μg

• Substrate: 10 μM

Background: MMP-12

Matrix metalloproteinases (MMPs) are a family of zinc and calcium dependent endopeptidases that collectively degrade the components of the extracellular matrix. MMP-12 (macrophage elastase) consists of the following domains: a pro domain, a catalytic domain containing the zinc-binding site, and a C-terminal hemopexin-like domain. The 52 kDa rmMMP-12 pro form is activated via processing into 45 kDa and 22 kDa active forms (1). MMP-12 is capable of cleaving several substrates in addition to elastin. Macrophage secretion of MMP-12 at sites of inflammation can be induced by cytokines. Given the secretion of MMP12 during an inflammatory response, MMP-12 is involved in many pathologies including vascular disease (2, 3) and cancer (4-6). In particular, MMP12-mediated pathological degradation of the extracellular matrix is a well-established key event in inflammatory-related pulmonary disease (7). For example, overexpression of MMP12 in alveolar macrophages is associated with smoking and emphysema (8) while different MMP-12 variants result in protection against chronic obstructive pulmonary disease (COPD) development (9) or cause increased risk and disease severity (10). Recently, MMP-12 has been shown to translocate into the nucleus of viral-infected cells to directly regulate transcription during an immune response (11).

• References:

1. Shapiro, S.D. et al. (1993) J. Biol. Chem. 268:23824.

2. Liu, S.L. et al. (2015) Sci. Rep. 5:17189.

3. Iyer, R.P. et al. (2015) Int. J. Cardiol. 185:198.

4. Cao, W. et al. (2017) Oncol. Rep. 3:1401.

5. Klupp, F. et al. (2016) BMC Cancer 16:494.

6. Chung, I.C. et al. (2014) BMC Cancer 14:348.

7. Vanderbroucke, R.E. et al. (2011) Eur. Respir. J. 387:1200.

8. Babusyte, A. et al. (2007) Respir. Res. 8:81.

9. Hunninghake, G.M. et al. (2009) N. Engl. J. Med. 361:2599.

10. Mukhopadhyay, S. et al. (2010) J. Allergy Clin. Immunol. 126:70.

11. Marchant, D.J. et al. (2013) Nature Medicine 20:493.

• Long Name:

  Matrix Metalloproteinase 12

• Entrez Gene IDs:

  4321 (Human); 17381 (Mouse)

• Alternate Names:

  EC 3.4.24; hME; HMEEC 3.4.24.65; Macrophage elastase; macrophage metalloelastase; matrix metallopeptidase 12 (macrophage elastase); matrix metalloproteinase 12 (macrophage elastase); Matrix metalloproteinase-12; ME; MGC138506; MME; MMP12; MMP-12