• 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 the fluorogenic peptide substrate, Mca-PLGL-Dpa-AR-NH    ₂ (Catalog # ). The specific activity is >500 pmol/min/μg, as measured under the described conditions.

• Source

  Mouse myeloma cell line, NS0-derived Leu17-Cys470, with Val97Leu substitution

• Accession #

• N-terminal Sequence    
  Analysis

  Leu17

• Structure / Form

  Pro form

• Predicted Molecular Mass

  52 kDa

• SDS-PAGE

  53-60 kDa, reducing conditions

Assay Procedure

Materials

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

• Recombinant Human MMP-12 (rhMMP-12) (Catalog # 917-MPB)

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

• Substrate MCA-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: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhMMP-12 to 50 μg/mL in Assay Buffer.

2. Activate rhMMP-12 by adding APMA to a final concentration of 1 mM and incubating at 37 °C for 24 hours.

3. Dilute activated rhMMP-12 to 0.4 ng/μL in Assay Buffer.

4. Dilute Substrate to 20 μM in Assay Buffer.

5. Load into plate 50 μL of 0.4 ng/μL rhMMP-12, and start the reaction by adding 50 μL of 20 μM Substrate.

6. Include a Substrate Blank containing 50 μL Assay Buffer and 50 μL of 20 μM Substrate.

7. Read at excitation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes.
   Calculate specific activity:
    
   

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

Per Well:

• rhMMP-12: 0.020 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 54 kDa rhMMP-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). MMP-12 has also 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