Recombinant Mouse MMP-12 Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse MMP-12 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a fluorogenic peptide substrate Mca-KPLGL-Dpa-AR-NH₂ (Catalog # ES010). The specific activity is >100 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse MMP-12 protein
  Ala18-Cys462
• Accession #: EDL24933
• N-terminal Sequence: Ala18
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 52 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 50-62 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in MES, NaCl, CaCl₂, CHAPS, ZnSO₄ and PEG.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Reconstitution Instructions: Reconstitute at 0.25 mg/mL in 10 mM MES, 0.1 M NaCl, 100 µM CaCl2, 0.1% CHAPS, 1 µM ZnSO4 and 0.1% PEG, pH 6.0
• Assay Procedure:
  • 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 # ES010), 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:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

  *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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse MMP-12 Protein, CF

• 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

Background

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).

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.

Publications for MMP-12 (3467-MPB)(4)

Publications using 3467-MPB

• Levengood, MR;Carosino, CM;Zhang, X;Lucas, S;Ortiz, DJ;Westendorf, L;Chin, AP;Martin, AD;Wong, A;Hengel, SM;Sun, H;Zeng, W;Yumul, R;Dominguez, MM;Chen, Y;Zheng, JH;Karlsson, CAB;Trang, VH;Senter, PD;Gardai, SJ; Preclinical development of SGN-CD47M: Protease-activated antibody technology enables selective tumor targeting of the innate immune checkpoint receptor CD47 Molecular cancer therapeutics 2024-10-28 [PMID: 39463068] (Bioassay, N/A)
• L Jiang, M Yang, S He, Z Li, H Li, T Niu, D Xie, Y Mei, X He, L Wei, P Huang, M Huang, R Zhang, L Wang, J Li MMP12 knockout prevents weight and muscle loss in tumor-bearing mice BMC Cancer, 2021-12-04;21(1):1297. 2021-12-04 [PMID: 34863141] (Bioassay, Mass Spectrometry, N/A)
• PM Andrault, P Panwar, NCW Mackenzie, D Brömme Elastolytic activity of cysteine cathepsins K, S, and V promotes vascular calcification Sci Rep, 2019-07-04;9(1):9682. 2019-07-04 [PMID: 31273243] (Cell Culture, Mouse)
• Anna K Kopec Fibri(nogen) drives repair after acetaminophen-induced liver injury via leukocyte ?M?2 integrin-dependent upregulation of MMP12 J. Hepatol, 2016-12-10;0(0):. 2016-12-10 [PMID: 27965156] (In Vivo, Mouse)

Bioinformatics

• Uniprot:
  • EDL24933()